Mycologia
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DOI: 10.3852/mycologia.97.6.1330
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Mycologia, 97(6), 2005, pp. 1330-1334.
© 2005 by The Mycological Society of America

Morphology of the rust fungus Puccinia boroniae revisited


S.A. Driessen 1
P.A. O’Brien
G.E St J. Hardy 2

     School of Biological Sciences and Biotechnology, Murdoch University, 3 South Street, Murdoch, Perth, Western Australia, Australia 6150


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 TAXONOMY
 LITERATURE CITED
 

Puccinia boroniae Henns. is a rust fungus endemic to Australia, infecting various Boronia spp. This study describes and illustrates, using light and scanning electron microscopy, the telial stage, teliospore germination and basidiospore production of specimens collected from commercial Boronia plantations in Western Australia. Unusual formation of a single basidiospore per germinating teliospore, and the pycnial stage, observed on Boronia megastigma leaves, are reported for the first time for P. boroniae.

Key words: basidiospores, Boronia, leptosporic, microcyclic, monokaryotic haustoria, pycnia, Rutaceae, teliospore germination, Uredinales


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 TAXONOMY
 LITERATURE CITED
 
Puccinia boroniae Henns. (Uredinales) is an endemic rust fungus of Australia, with its known host range including several species of Boronia, a native Australian wildflower (Sampson and Walker 1982Go, Cook and Dubae 1989Go, Shivas 1989Go). In southwestern Western Australia Boronia is cultivated commercially, with harvested stems primarily destined for export cut-flower markets (Lidbetter and Plummer 2004Go). Several of the more commonly cultivated species and varieties, such as Boronia heterophylla and B. megastigma, are susceptible to P. boroniae. Infected plants exhibit telial pustules on stems and leaves, with defoliation of infected leaves eventually occurring. Although the disease does not directly result in plant death, the presence of rust in a plantation drastically reduces the number of harvestable stems, resulting in economic loss to the growers.

P. boroniae was described by several authors in the beginning of the 20th century (Hennings 1903Go, Sydow and Sydow 1904Go, McAlpine 1906Go). All descriptions are similar, recording only the telial stage (teliospores and mesospores, and the gross morphology of the telium) of the rust fungus from one specimen (on branches of Boronia spinescens Benth. from Western Australia, collected by L. Diels). The records also described P. boroniae as a Leptopuccinia, a microcyclic rust fungus in which mature teliospores germinate without a period of dormancy. However descriptions of the germination structures and basidiospores were not recorded. No modern descriptions of P. boroniae have been published subsequent to these, a likely reflection of the low economic importance of Boronia before its extensive commercial cultivation. The objectives of this study were to provide a current and more detailed morphological description of the various spore stages of P. boroniae observed in the field and to describe the mode of teliospore germination and basidiospore formation. Classification of P. boroniae as a microcyclic rust fungus was assessed through controlled inoculation trials.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
TAXONOMY
 LITERATURE CITED
 
Fortnightly to monthly examination of B. heterophylla, B. megastigma and other susceptible species and varieties of Boronia grown on commercial plantations in the great southern region of Western Australia were made Feb 2003–Jul 2004. Fresh specimens were obtained for spore and sorus examinations, as well as teliospore germination. Herbarium specimens of P. boroniae collected from within the same region also were examined.

Sorus morphology.— – Color designation of the various sorus stages was made from Kornerup and Wanscher (1967)Go. Telia, pycnia and the intra/intercellular fungal structures were observed in cleared and stained, stained only and unstained hand sections of fresh leaf and stem material. Cleared sections were prepared using a modified version of the methods of Quilliam and Shattock (2003)Go. Stained only and unstained sections were mounted directly in 0.05% lactoglycerol cotton blue and lactoglycerol, respectively. Sections were viewed under oil at 1000x magnification with an Olympus BH-2 microscope with bright field and differential interference contrast (DIC) and photographed with an Olympus DP10 digital camera. Images were edited for clarity where necessary with Adobe Photoshop® 7.0.

The surface morphology of the telia on leaves and stems was examined by scanning electron microscopy (SEM). Several telial samples were hand-sectioned through the middle of the telium before fixation. Specimens were fixed overnight at 4 C in 3% glutaraldehyde in 0.025M phosphate buffer (pH 7.0), washed several times in buffer, dehydrated in a graded series of ethanol, with a final wash of amyl acetate. Each specimen was critical point dried, adhered to an aluminium stub with carbon paste, sputter-coated with gold in a Balzers Union SCD 020 (Balzers Union Ltd, Liechtenstein) and examined under a Philips XL20 scanning electron microscope at 5–10 kV.

Spore morphology.— – Teliospores and pycniospores were mounted in lactoglycerol, gently heated to expand collapsed spores and examined by bright field and DIC microscopy as described previously. Spore dimensions were determined with Olysia BioReport Imaging Software version 3.2 (Olympus, Australia) and digital images recorded with an Olympus BX51 microscope attached to a MicroPublisher 3.3 RTV photographic unit (Olympus, Australia).

Teliospore germination.— – Freshly collected leaves bearing telia were soaked 1–4 h in sterile distilled water at 15 C in the dark and blotted dry with sterile filter paper to remove excess water. Intact telia retained on the host leaves were incubated in sealed Petri dishes lined with moist filter paper and incubated at 15 and 20 C in the dark. Preliminary studies had shown that teliospore germination and basidiospore formation was optimal at 15–20 C in the dark (Driessen unpublished). Germination also was assessed on whole telia detached from the underling plant material under a dissecting microscope by placing detached telia upright onto 2% distilled water agar plates and incubating at 10, 15, 20 and 25 C in the dark. Telia were examined for the presence of germination structures and basidiospores at hourly intervals for 8 h at 400x with bright field and DIC microscopy. After 24 h germinating teliospores were gently teased from each sorus with a fine needle, mounted in lactoglycerol and examined as previously described.

Host inoculation.— – Several branches from a heavily infected B. heterophylla plant were soaked 2 h in sterile distilled water at 20 C in the dark. The inoculum was blotted dry with sterile filter paper and placed among the top branches of mature healthy B. heterophylla plants (18–24 mo old) to allow for natural dispersal of the basidiospores produced from the intact telia (Morin et al 1993Go). Inoculated plants were gently misted with water, covered with thick opaque plastic bags to ensure high humidity was maintained and placed in a controlled growth cabinet maintained at 20 ± 1 C with a 12 h photoperiod. Plants were misted daily for 2 d after which the inoculum and plastic bags were removed, and inoculated plants then were checked daily for signs of infection. The removed inoculum was examined immediately under a dissecting microscope to ensure that teliospore germination and basidiospore formation had occurred.


   TAXONOMY
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 TAXONOMY
LITERATURE CITED
 
Puccinia boroniae Henns. Hedwigia 42:73. 1903.

FIGS. 1Go–16GoGoGoGoGo


Figure 1
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  		FIGS. 1–3. Telia of Puccinia boroniae. 1. Confluent telia (arrow) along stem of Boronia megastigma. Bar = 5 mm. 2. Mature telia on adaxial surface of Boronia heterophylla leaf. Arrow indicates a telium not yet erupted through the leaf epidermis. Bar = 1 mm. 3. Semimature telium surrounded by ruptured epidermis of the adaxial surface of B. megastigma leaf with mature (pigmented) teliospores visible. Bar = 0.2 mm.

 

Figure 2
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  		FIG. 4. SEM of cross section of a telium, showing mature teliospores just beneath the leaf epidermis (arrow). E = epidermal cell. Mc = mesophyll cell(s). Bar = 50 µm.

 

Figure 3
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  		FIGS. 5–9. Teliospores and mesospores of Puccinia boroniae. 5 and 6. Two and three-celled teliospores of P. boroniae with apical germ pore (arrow) visible. 7 and 8. Variation in septum formation in teliospores. M = mesospore; IM = immature (primary) teliospore. 9. Mesospores. Bars = 10 µm.

 

Figure 4
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  		FIGS. 10, 11. M-haustoria of Puccinia boroniae in mesophyll cells beneath a telium. Note constricted neckband region at point of entry into cell (arrow). Bar = 10 µm.

 

Figure 5
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  		FIGS. 12, 13. Teliospore germination structures and basidiospores of Puccinia boroniae. Bs = basidiospore; G = germ tube; Mb = metabasidium; St = sterigma. Bar = 20 µm.

 

Figure 6
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  		FIGS. 14–16. Pycnial stage of Puccinia boroniae on Boronia megastigma. 14. Honey-like fluid being released from pycnia (arrow). Bar = 1 mm. 15. Type 4 pycnial structure of P. boroniae with released pycniospores present near ostiole (arrow). Bar = 100 µm. 16. Subepidermal pycnial primordia showing focused growth towards stomatal opening. SO = stomatal opening; MC = mesophyll cell. Bar = 40 µm.

 
Uredia and aecia not observed. Telia erumpent and pulvinate, amphigenous on leaves, stems, peduncles and sepals, reddish brown (9E7-8) to dark brown (9F6-8), mostly scattered or moderately concentric, individually up to 2.5 mm wide, often confluent on stems and peduncles, subepidermal, in the leaf arising between the mesophyll and epidermis, and in the stem between the cortex and epidermis from a hyaline, dense pseudoparenchymatous layer up to 20 µm thick, composed of globose to angular hyaline cells, 4–6 µm diam, giving rise to aseptate, hyaline, cuboidal to rectangular teliospore initials. Teliospore initials developing a single horizontal septum, forming the pedicel and primary teliospore, initially rectangular, unicellular and hyaline, broadening with maturity to become more ellipsoid, two-celled and pigmented, forming a compact, erumpent mass of teliospores. Teliospores predominantly 2-celled, with a single, horizontal, occasionally oblique or vertical septum, rarely 3-celled or 4-celled, slightly constricted at septum, cinnamon to dark brown, smooth, pedicellate, broadly ellipsoidal, (22–)24–35(–37) x (13–)14–19(–20) µm (average 28.8 ± 2.8 x 16.6 ± 1.4 µm; n = 235), a single germ pore observed in each cell, apical in upper cell and septal in lower cell, germinating without dormancy. Mesospores ellipsoid to obovoid with single apical germ pore, coloration similar to teliospores, 18–29(–32) x 13–18(–20) µm (average 23.5 ± 3.1 x 15.7 ± 1.9 µm; n = 99). Pedicel persistent, hyaline to pale yellow, up to 130 µm long, attached at bottom of basal cell of mesospores and teliospores, occasionally obliquely or laterally inserted. Haustoria coiled and branched, with a well defined neckband at the entry point into host mesophyll cells. Basidiospores formed on single sterigma at subterminal end of the metabasidium, ovate to elliptical with a prominent apiculus, 13.5–18.6 x 8.8–11.7 µm (average 16.4 ± 1.0 x 10.5 ± 0.6 µm; n = 80). Pycnia amphigenous, arranged in small clusters, individually up to 350 µm wide, yellowish-orange (4A7-8) to orange (6A/B8), subepidermal, arising from an extensive network of intercellular hyphae within host tissue, aggregating beneath stomata to form pycnial primordials, mature pycnium ampulliform with numerous straight, unbranched, spine-like periphyses present above the hymenium and just below the ostiole, hymenium consisting of a layer of pseudoparenchymatous cells giving rise to long, slender pycniosporophores up to 30 µm long. Pycniospores subpyriform to ellipsoidal, hyaline, smooth, 2.8–5.0 x 1.6–2.6 µm (average 3.8 ± 0.5 x 2.0 ± 0.2 µm; n = 80), released in a honey-like fluid through the ostiole of the pycnium.

Specimens examined. – AUSTRALIA. WESTERN AUSTRALIA: Albany, commercial nursery, (35°01'S, 117°50'E), Boronia heterophylla, Jul 2004, S.A. Driessen (WAC 12425); Mount Barker, private flower plantation, (34°34'S, 117°46'E), Boronia megastigma, May 2000, S.A. Driessen (WAC 12424); same local, Boronia megastigma, Feb 2004, S.A. Driessen (WAC 12426); Redmond, private flower plantation, (34°54'S, 117°33'E), Boronia clavata, Jun 2000, S.A. Driessen (WAC 12427); same local, Boronia heterophylla, Jun 2000, S.A. Driessen (WAC 12428).

Commentary. – Examination of the specimens in this study showed that telia predominantly formed on leaves in a scattered arrangement. Although telia also were observed on stems in a number of specimens in this study, we observed that extensive confluent formation of telia on the stems, as described by Hennings (1903)Go, Sydow (1904)Go and McAlpine (1906)Go, was restricted to specimens from B. megastigma (WAC 12426). Telium structure of P. boroniae was typical of many Puccinia spp., being subepidermal, erumpent, with a well developed basal layer of sporogenous cells from which teliospores were born singly on pedicles (Cummins and Hiratsuka 1983Go, Mendgen 1984Go). Morphology of the teliospores agreed with previous descriptions, although a greater range of mesospore dimensions were recorded in this study in comparison to those reported by McAlpine (1906)Go (ca. 30–34 µm x 17–18 µm).

The confinement of inter- and intracellular hyphae to the host mesophyll layers indicated a nonsystemic mode of infection. The observation of M-haustoria, the formation of which are associated with infections by basidiospores in rust species (Littlefield 1981Go, Quilliam and Shattock 2003Go), suggested a microcyclic lifecycle, in which telia develop from successful basidiospore infection (with or without pycnia production) without the intermediates of aeciospores or urediospores, which have never been observed on infected hosts in the field.

Teliospores of P. boroniae germinated immediately without a period of dormancy, confirming the description provided by earlier authors (Hennings 1903Go, Sydow and Sydow 1904Go, McAlpine 1906Go). Consistent and reproducible formation of a single basidiospore from each metabasidium was observed under all experimental conditions, indicating this to be the normal behavior of the rust pathogen. A whip-like germ tube occasionally formed on the sterigma instead of a basidiospore. This abnormal germination structure was associated with germinating teliospores that remained submerged in excessive water, an occurrence reported by other authors (Gardner 1996Go, Ono 2002aGo). Several authors (Peterson 1974Go, Hiratsuka and Sato 1982Go, Ono 2002bGo) have reviewed the diversity of basidial development of rust fungi, and though the formation of a single basidiospore is unusual, it previously has been reported for Puccinia rutainsulara (Gardner 1994Go) and Uromyces alyxiae (Gardner 1987Go).

The pycnial stage of P. boroniae was detected on a single host plant species (B. megastigma) at a single commercial plantation in Mount Barker, Western Australia. The structure of the pycnium conformed to type 4 as described by Hiratsuka and Cummins (1963)Go, typical of Puccinia spp. and characterized by determinate growth, subepidermal and strongly convex hymenia, with well developed bounding structures (periphyses). Accompanying the periphyses with acute apical tips were hyphae that were blunt at the distal end, presumed to be flexuous hyphae. However these were morphologically difficult to distinguish with the techniques employed. Unlike other Puccinia spp., such as P. recondita (Gold et al 1979Go), ostiole formation was seen to occur by the extension of periphyses through stomatal openings as reported for the rust fungus Melampsora lini (Gold and Littlefield 1979Go), rather than rupturing through the leaf epidermis. Because the material examined was naturally infected rather than inoculated with a single basidiospore, it was impossible to determine whether the cluster of multiple pycnia observed on each leaf surface developed from a single basidiospore or from multiple spores. Although pycnia were present on leaves of many B. megastigma plants within this plantation, many infected plant parts also exhibited only telia at varying stages of maturity. Furthermore no other specimens of P. boroniae collected from different locations exhibited the pycnial stage despite extensive sampling. This would suggest that the pycnial stage may not be a prerequisite for the development of the telial stage and, although still present, may be nonfunctional in the rust lifecycle.

Inoculation of B. heterophylla with basidiospores of P. boroniae resulted in development of telia on the leaves within 21 d, confirming the lifecycle as microcyclic as suggested by Hennings (1903)Go and McAlpine (1906)Go. Small yellow discolorations initially were detected on the leaf surface 15–17 d after inoculation, developing into mature telia that ruptured through the leaf epidermis within 3–5 d. Once teliospores had ruptured the leaf surface, they were mature enough to germinate and produce basidiospores, assessed by incubating leaves bearing telia in a moist chamber after 1 h exposure to water. Basidiospore formation was observed within 3–6 h. No pycnia were observed on the inoculated B. heterophylla plants. However the specimen exhibiting pycnia in the field was not employed in the trial due to decreased viability of the teliospores, and whether the pycnial stage is functional in the lifecycle of P. boroniae remains inconclusive.


   ACKNOWLEDGMENTS
 
Financial support for this research was provided by the Western Australian Department of Agriculture, as the industry partner for ARC LINKAGE APAI grant C00107300. Gordon Thomson, Murdoch University, is acknowledged for his invaluable technical support and informative discussions relating to the histological and SEM specimen preparation. The authors thank Dr Paul Barber and Dr Treena Burgess for their critical review of the manuscript.


   FOOTNOTES
 
Accepted for publication June 26, 2005.

1 Current address: NSW Department of Primary Industries, Locked Bag 1000, Narrabri, NSW 2390 Australia Back

2 Corresponding author. E-mail: g-hardy{at}murdoch.edu.au


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 TAXONOMY
 LITERATURE CITED
 
Cook RP, Dubae AJ. 1989. Host-pathogen index of plant diseases in South Australia. Adelaide: South Australian Department of Agriculture. 142 p.

Cummins GB, Hiratsuka Y. 1983. Illustrated genera of rust fungi. Minnesota: American Phytopathological Society Press. 152 p.

Gardner DE. 1987. Teliospore germination of Uromyces alyxiae, an endemic Hawaiian rust. Mycologia 79:914–917.[CrossRef]

———. 1994. Teliospore germination and nuclear behavior of Puccinia rutainsulara, a microcyclic Hawaiian rust. Mycologia 86:486–493.[CrossRef]

———. 1996. Puccinia rugispora: an unusual microcyclic rust endemic to Hawaii. Mycologia 88:671–676.[CrossRef]

Gold RE, Littlefield LJ. 1979. Light and scanning electron microscopy of the telial. pycnial, and aecial stages of Melampsora lini. Can J Bot 57:629–638.[CrossRef]

———, ———, Statler GD. 1979. Ultrastructure of the pycnial and aecial stages of Puccinia recondita. Can J Bot 57:74–86.

Hennings P. 1903. Puccinia boroniae. Hedwigia 42:73.

Hiratsuka Y, Cummins GB. 1963. Morphology of the permogonia of the rust fungi. Mycologia 55:487–507.[CrossRef]

———, Sato S. 1982. Morphology and taxonomy of rust fungi. In: Scott KJ, Chakravorty AK, eds. The Rust Fungi. London: Academic Press. p 1–36.

Kornerup A, Wanscher JH. 1967. Methuen handbook of colour. 2nd ed. London: Methuen & Co Ltd. 243 p.

Lidbetter J, Plummer J. 2004. Boronia. In: Salvin S, Bourke M, Byrne T, eds. The new crop industries handbook. RIRDC Publication No. 04/125. Canberra, Australia: Rural Industries and Research Corp. p 420–427.

Littlefield LJ. 1981. Biology of the Plant Rusts: an introduction. Ames: Iowa State University Press. 103 p.

McAlpine D. 1906. The Rusts of Australia: their structure, nature and classification. Melbourne, Australia: Melbourne Government Printer (Dept. of Agriculture). p 181, pl. XIII.

Mendgen K. 1984. Development and physiology of teliospores. In: Bushnell WR, Roelfs AP, eds. The cereal rusts. London: Academic Press Inc. p 375–398.

Morin L, Auld BA, Brown JF. 1993. Host range of Puccinia xanthii and postpenetration development on Xanthium occidentale. Can J Bot 71:959–965.

Ono Y. 2002a. Life cycle and nuclear behaviour in three rust fungi (Uredinales). Mycoscience 43:37–45.[CrossRef]

———. 2002b. The diversity of nuclear cycle in microcyclic rust fungi (Uredinales) and its ecological and evolutionary implications. Mycoscience 43:421–439.[CrossRef]

Peterson RH. 1974. The rust fungus life cycle. Bot Rev 40:453–513.[CrossRef]

Quilliam RS, Shattock RC. 2003. Haustoria of microcyclic rust fungi Uromyces ficariae and Puccinia tumida and other gall-forming species, U. dactylidis (macrocyclic) and P. smyrnii (demicyclic). Plant Pathol 52:104–113.[CrossRef]

Sampson PJ, Walker J. 1982. An annotated list of plant diseases in Tasmania. Hobart: Department of Agriculture Tasmania. 121 p.

Shivas R. 1989. Fungal and bacterial pathogens of plants in Western Australia. J of Royal Soc Western Australia 72:1–62.

Sydow P, Sydow H. 1904. Puccinia boroniae. Monographia Uredinearum. p 891–892, pl. XLV.





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