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South African Journal of 2005, 71(3&4): 307–311 Copyright © NISC Pty Ltd Printed in — All rights reserved SOUTH AFRICAN JOURNAL OF BOTANY EISSN 1727–9321

Seasonal trends in colonisation of infructescences by Gondwana- myces and Ophiostoma spp.

F Roets1, LL Dreyer1* and PW Crous2

1 Department of Botany, University of , Private Bag X1, Matieland 7602, South Africa 2 Centraalbureau voor Schimmelcultures, Fungal Biodiversity Centre, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands * Corresponding author, e-mail: [email protected]

Received 6 April 2004, accepted in revised form 18 November 2004

Seasonal growth of the fungal genera Gondwanamyces of Protea. A definite seasonal pattern was observed, with and Ophiostoma (hereafter referred to as ophiosto- colonisation numbers peaking during the wetter winter matoid fungi) on the floral parts of serotinous Protea- months. P. laurifolia was found to be a new host for ceae flowers was investigated. Several new Protea host Ophiostoma splendens and Gondwanamyces capensis. were found and new knowledge emerged Ophiostomatoid fungi were restricted to dead floral parts, regarding the tissue types colonised by these fungi. and fruiting structures were never observed on living Although floral parts of a wide range of were tissue. Both the vector organisms and the specific examined, ophiostomatoid fungi were exclusively ecological function of the ophiostomatoid fungi are still collected from the infructescences of serotinous species unknown, and require further investigation.

Introduction

The floral diversity of the (CFR) is world- two genera, namely Ophiostoma H. Syd. and P. Syd., renowned. This area includes the Biome, which including the species Ophiostoma protearum Marais and contains most of the c. 9 000 plant species (Goldblatt and Wingf., O. splendens Marais and Wingf. and O. africanum Manning 2000) found within the CFR. The fynbos contains Marais and Wingf. (Sporothrix Hektoen and Perkins three dominant plant families: Ericaceae, and anamorphs), and Gondwanamyces Marais and Wingf. Proteaceae (Cowling and Richardson 1995), of which the including the species Gondwanamyces capensis (M.J. Proteaceae is often the structurally dominant member. Wingf. and P.S. van Wyk) Marais and Wingf., and G. The Proteaceae is diverse in terms of number of species proteae (M.J. Wingf., P.S. van Wyk and Marasas) Marais and the range of morphological forms that exist within the and Wingf. (Knoxdaviesia Wingf. et al. anamorphs). These family. In the south- alone, there are more than two ascomycete genera share the same morphological 330 species in 14 genera (Rebelo 1995). In total, 13 of these characteristics of spherical ascocarps with long necks, but genera are Cape-centred, with 10 endemic to the area (Rourke differ in their anamorphs (Wingfield and Van Wyk 1993) 1998). The Proteaceae is the seventh largest family of vascular and cycloheximide tolerance (Marais 1996). in the CFR, with 96.7% of its members confined to this Morphologically similar genera with similar long perithecial area (Goldblatt and Manning 2000). Protea L. species are of necks from the Northern Hemisphere are insect-vectored considerable economic importance to South Africa in terms of (Davidson and Robinson-Jeffrey 1965, Davidson et al. 1967, eco-tourism, horticulture and the dried-flower industry. Davidson 1978, Dowding 1984), and cause important tree Several fungi that are closely associated with the Protea- diseases such as Dutch elm disease (Braisier 1988) and ceae have been discovered and described (Crous et al. 2000a, oak wilt (Sinclair et al. 1987). Some authors have suggested 2000b, Swart et al. 2000, Taylor and Crous 2000, Taylor 2001). that these fungi may also assist bark beetles to overwhelm Many of these cause fungal infections, including diseases of host tree resistance (Christiansen and Solheim 1990), the , stems, roots and seedlings (Crous et al. 2000a, hinting at the existence of mutualistic relationships between 2000b). Very limited attention has, however, been focussed the fungi and the insect vectors. on the apparently non-pathogenic fungi associated with these There is a strong morphological similarity between Northern plants (Marais and Wingfield 1994, Taylor et al. 2001). Hemisphere Ophiostoma species that occur in the galleries Five ophiostomatoid fungal species are currently known of bark beetles on trees (Wingfield et al. 1999) and the to inhabit the infructescences of some serotinous Protea Ophiostoma and Gondwanamyces species present in Protea species, where they are thought to grow as saprobes (Marais infructescences. The morphological similarities revolve around and Wingfield 2001). These ophiostomatoid fungal the flask-shaped ascomata with long necks that are present species are only associated with the infructescences of in both groups. This morphological arrangement suggests Protea species from South Africa. They are grouped into insect spore dispersal. Spores collect in a sticky mass at the 308 Roets, Dreyer and Crous tips of the necks, where insects can readily come into contact Proteaceae were collected from various sites in the Stellen- with them (Upadhyay 1981, Wingfield et al. 1993). bosch region, South Africa (Table 1). Species were selected These morphological similarities are, to some extent, according to the availability of their infructescences in the corroborated by an rDNA-based molecular phylogeny of the region. Their infructescences were examined for the ophiostomatoid fungi by Marais et al. (1998). In this phylogeny, presence of ophiostomatoid perithecia and their anamorphs, the southern hemisphere members of Ophiostoma form a using a dissecting microscope (X100 magnification). This well-supported monophyletic group sister to the species enabled the identification of the ideal infructescence age for of Ophiostoma, O. piliferum (Fr.) Syd. and P. Syd., which is detecting fungal growth. Ten fruiting structures (c. one year a Northern Hemisphere representative. The southern African old) of each of these species were then collected at each members of the genera Gondwanamyces and Ophiostoma, site (30 samples in total for each species) at three-month however, are clearly paraphyletic, which suggest that the intervals (from February 2000 to November 2001) and morphological similarities between these two genera must be inspected for the presence of ophiostomatoid fungal fruiting the result of convergent evolution (Wingfield et al. 1999). structures. Fungi were identified directly from the host To date, ophiostomatoid fungi in Protea species have only material and from isolations done on Petri dishes containing been found in insect-infested flower heads (Wingfield et al. 2% malt extract agar (MEA; Biolab, Midrand, South Africa) 1988), suggesting that one or more of these insects could and Sigma Streptomycin sulphate (0.04g l–1). act as vector of the fungal spores. Insects associated with In addition, a selection of other Proteaceae present at the Protea infructescences belong to a wide range of families study sites, the Betty’s Bay/ area and the Cape (Coetzee and Giliomee 1987a, 1987b, Coetzee 1989, Point Nature Reserve (Western Cape Province), were Wright 1990, Visser 1992). The putative vectors of G. screened for the presence of ophiostomatoid fungi in their proteae have, however, been narrowed down to only two flower heads at different times of the year. These included species, and the vectors for O. splendens have been several Protea species (P. compacta R. Br., P. lepido- narrowed down to four species (Roets 2002). Further carpodendron (L.) L., P. longifolia Andrews, P. magnifica Link, molecular studies are underway to identify the vectors of P. grandiceps Tratt, P. scabra R. Br. and P. speciosa (L.) L.), each of the southern African ophiostomatoid species. a number of R. Br. species (L. conocar The aim of the present study was to examine the seasonal podendron (L.) H. Beuk, L. oleifolium (P.J. Bergius) R. Br. growth patterns of ophiostomatoid fungi within Protea and L. cordifolium (Salisb. ex Knight) Fourc.), two infructescences, while also scanning this niche for new R. Br. species (L. laureolum (Lam.) Fourc. fungal and host associations. The nature and extent of and L. xanthoconus (Knutze) K. Schum.), ophiostomatoid colonisation were also investigated. thymelaeoides (P.J. Bergius) Rourke, cucullatus (L.) R. Br., umbellata (Thunb.) R. Br. and Materials and Methods curvifolia Salisb. ex Knight. These plants were not included in the three-monthly monitoring due to the limited numbers Seasonality and host specificity of available flower heads they presented. They were merely included to help determine the host range of the different Six-month- to one-year-old infructescences and other fruiting fungal species. structures (e.g. cones) of 11 species belonging to the

Table 1: Collection sites of Proteaceae infructescences and other fruiting structures in the Stellenbosch region of South Africa

Proteaceae species Population site Grid reference Leucadendron rubrum Burm. f. Jonkershoek S: 33° 58.591' E: 18° 56.817' Leucadendron rubrum Stellenbosch Mountain S: 33° 56.743' E: 18° 52.711' Bergius Jonkershoek S: 33° 59.210' E: 18° 57.361' Leucadendron salignum Stellenbosch Mountain S: 33° 56.743' E: 18° 52.711' L. Jonkershoek S: 33° 59.210' E: 18° 57.361' Stapf. Stellenbosch Mountain S: 33° 56.743' E: 18° 52.711' Protea burchellii Jan S Marais Park S: 33° 55.984' E: 18° 52.375' Protea burchellii Jonkershoek S: 33° 58.591' E: 18° 56.817' L. Jan S Marais Park S: 33° 55.984' E: 18° 52.375' Fourc. Jan S Marais Park S: 33° 55.984' E: 18° 52.375' Thunb. Stellenbosch Mountain S: 33° 56.743' E: 18° 52.716' Protea laurifolia Franschoek pass S: 33° 54.354' E: 19° 09.454' Protea laurifolia Jan S Marais Park S: 33° 55.984' E: 18° 52.375' R. Br. Jan S Marais Park S: 33° 55.984' E: 18° 52.375' Protea neriifolia Franschoek pass S: 33° 54.448' E: 19° 10.096' Protea neriifolia Jonkershoek S: 33° 59.555' E: 18° 58.287' Mill. Jonkershoek S: 33° 59.210' E: 18° 57.361' L. Jan S Marais Park S: 33° 55.984' E: 18° 52.375' Protea repens Stellenbosch Mountain S: 33° 56.743' E: 18° 52.711' Protea repens Jonkershoek S: 33° 59.555' E: 18° 58.287' fasciflora Salisb. ex Knight Jonkershoek S: 33° 58.591' E: 18° 56.817' South African Journal of Botany 2005, 71(3&4): 307–311 309

Statistical analysis insect vectors, indicates that insect vectors play a significant role in the dissemination of spores of fungi inhabiting Presence or absence of ophiostomatoid fruiting structures Proteaceae infructescences. in infructescences of the different Protea spp. (three sites per species) were recorded and represented as a Host tissue colonisation percentage (out of 10 infructescences sampled per plant species per site), and the percentage data were arcsine- Perithecia and conidiophores were found only on senescent transformed (Zar 1984). The influence of the factors and plant tissues. Fungi were confined to the dead styles and their interactions were tested with a one-way analysis of other dead floral parts of the individual flowers within the variance (ANOVA) (Statgraphics Version 7, 1993, Stat- host infructescences. In highly colonised infructescences, graphics Corporation, USA). Where the ANOVA revealed some perithecia were also found growing on the inside of significant effects by the factors, the differences between involucral surrounding the infructescence. Perithecia samples were separated using a post hoc Fisher’s Protected and conidiophores were never observed growing on the LSD (P < 0.05). Different colours were used to indicate fruits of any of the Protea hosts. significant differences between samples. Seasonal growth patterns Fungal cultures The numbers of ophiostomatoid fungi that colonised Protea Fungal cultures were obtained by removing spores from the infructescences displayed distinct seasonal fluctuations (Figure apices of ascomatal necks or conidiophores with a sterile 1). There was a marked increase in the number of perithecia needle. These were plated onto agar surfaces in Petri and conidiophores in Protea infructescences during the wetter dishes containing 2% malt extract agar (MEA; Biolab, winter months (June–August), with colonisation percentages Midrand, South Africa) and Sigma Streptomycin sulphate (total number of infructescences containing ophiostomatoid (0.04g l–1). The isolates were then incubated at 21°C in the fruiting structures out of a sample size of 10) being as high dark for 7–9 days prior to fungal identification. as 80% in some cases (G. capensis on P. laurifolia at Stellenbosch Mountain site during winter collection). In many Determination of tissue colonisation instances this increase in incidence of the fungal perithecia and conidiophores was statistically significant (Figure 1). Light microscopy was used to study the colonisation sites and to scan for perithecia or conidiophores present on green Discussion (living), brown (dead without obvious ophiostomatoid colonisation) and senescent (dead with ophiostomatoid The results of Marais (1996) were corroborated by the colonisation) plant tissues. This facilitated an assessment results of the present study, in which ophiostomatoid fungi of the ability of the ophiostomatoid fungi to colonise the were found to colonise the infructescences of serotinous host Protea species in vivo. Protea species only. None of these fungi were found on the flower heads of any of the other Proteaceae genera, Results or on non-serotinous Protea species. To date, ophios- tomatoid fungi from the south-western Cape were only iso- Host specificity lated from Protea species belonging to the ‘true’ sugarbush, the bearded sugarbush and the spoon- sugarbush Ascomata and conidiophores of ophiostomatoid fungi were groups (Rebelo 1995). Representatives from other Protea exclusively found in infructescences of the Protea, sp. groups not included in this study should also be and were never detected on any of the other plant genera evaluated in future studies to elaborate on ophiostomatoid examined. These fungi were found to be prolific on P. host ranges. The factors that control host specificity are burchellii, P. laurifolia, P. neriifolia and P. repens infruc- unknown, but may include microclimatic conditions or tescences. No ophiostomatoid perithecia or conidiophores chemical differences between the different Protea species. were found in flower heads that were less than three months Different vectors for each of the fungal species may also old, while infructescences as old as five years still showed have a causal influence, as was sugges-ted by Marais and colonisation by ophiostomatoid fungi (e.g. G. proteae on P. Wingfield (1994). repens, collected on Stellenbosch Mountain). Two species of ophiostomatoid fungi were found to be A new ascomycete, recently described as Rhynchostoma prolific on P. laurifolia, namely O. splendens and G. proteae S. Lee and Crous, sp. nov. (Lee et al. 2003), was capensis. In both cases their presence on P. laurifolia repre- also isolated from P. laurifolia and P. burchellii. This fungus sents a new host record. Known host species for O. was found within the same ecological niche as the splendens now include P. laurifolia, P. lepidocarpodendron, ophiostomatoid fungi, infesting the pollen presenters. R. P. longifolia, P. neriifolia and P. repens (Marais and Wingfield proteae is morphologically similar to the ophiostomatoid 1994). Host species for G. capensis include P. laurifolia, P. fungi, and appears to share the same dispersal mechanism. burchellii, P. coronata, P. lepidocarpodendron, P. longifolia, Like the ophiostomatoid fungi, it produces sticky droplets P. magnifica and P. neriifolia (Wingfield and Van Wyk 1993, of spores at the apices of long perithecial necks, from where Marais and Wingfield 1994). Each of the remaining three insects can readily facilitate spore dispersal. The occurrence ophiostomatoid species are restricted to one species of of multiple fungal species thought to be associated with Protea only: G. proteae is exclusively found on P. repens 310 Roets, Dreyer and Crous

60 O. splendens on P. neriifolia overview of the host specificity of these fungi. The age of the youngest infructescence found to be colonised by ophiostomatoid fungi (three months old) 40 suggests that the vectors must be active after flowering to ensure fungal spore dispersal. Pollinators, such as bees, 20 can thus be excluded as possible vectors of these fungi as the flower heads of Protea species are closed by the time the fungi sporolate (Marais 1996). Not all flower visitors 80 G. capensis on P. laurifolia can, however, be excluded as vectors, as some insect borers (e.g. Genuchus hottentottus) complete their 60 immature stages in closed infructescences of various ages 40 (Coetzee and Giliomee 1987a). Once mature, they emerge from the infructescences, and could potentially come into 20 contact with ophiostomatoid spores. From here they could carry the spores to uncolonised of Protea species where they feed on pollen and . The fungi O. splendens on P. laurifolia 80 are retained in the inflorescences (as spores or as asexual stages), and only develop the sexual stage once the 60 inflorescences have matured to form closed infructescences. However, conidiophores of the anamorphs of Ophiostoma 40 and Gondwanomyces were never observed in isolation, and 20 were always accompanied by ascomata. Anamorphs were mostly observed in culture only.

INFRUCTESCENCES (%) 80 G. proteae on P. repens No ophiostomatoid fungi were found growing on the fruits of any of the colonised Protea infructescences, and no 60 perithecia were found penetrating the host tissue. This agrees with the suggestions by Marais (1996) that these 40 fungi may not have a direct influence on seed viability. They were also restricted to dead and senescent floral parts, 20 suggesting that the fungi have a saprobic, rather than a parasitic, relationship with their plant hosts. The fungi may, 80 G. capensis on P. burchellii however, influence the composition of the insect taxa that infest the infructescences, and in this way, could indirectly 60 influence seed production, viability and/or dispersal. When the ophiostomatoid fungi are found growing within Protea 40 infructescences, no other fungi are found to co-inhabit the 20 infructescences in significant numbers. In contrast, infruc- tescences that lack ophiostomatoid fungi are dominated by large numbers of different fungal species. This may be the result of the ophiostomatoid fungi out-competing other fungi once they become established within the flower heads. As suggested by Marais (1996), this competitiveness may protect 2000 Winter2000 Spring 2001 Winter2001 Spring 2000 Autumn 2000 Summer2001 Autumn 2001 Summer the Protea seeds from destruction by other, more destructive, fungal species. Further research is needed to examine these Figure 1: The mean percentage of Protea infructescences (10 fungal interactions and their impact on Protea seed viability. infructescences at each site) containing Ophiostoma and Gondwa- The ophiostomatoid fungi within the Protea infructe- namyces spp. ascomata and conidiophores over a two-year scences display distinct seasonal growth patterns. The peak collecting period (2000–2001) in the Stellenbosch district, South in fungal ascomata and conidiophores during autumn and Africa. Black bars indicate significant differences between seasons winter indicates that sufficient moisture (and perhaps also indicated by white/no bars (Fisher’s Protected LSD, P < 0.05) for low temperatures) could promote fungal growth. Differences the species, while grey bars indicate no significant difference found were observed in the seasonal distributions of ophio- to any other bars. Markers on bars indicate standard deviations from the mean stomatoid fungi in P. repens infructescences collected from Cape Point and Stellenbosch (+100km apart). Although these fungi were found to be prolific in infructescences (Wingfield et al. 1988), O. africanum is exclusively found collected at Cape Point during February, data from this on P. gaguedi (Marais and Wingfield 2001), and O. study show that very few infructescences contained protearum is exclusively found on P. caffra (Marais and ascomata and conidiophores during the same period at the Wingfield 1997). Apart from the Proteaceae, many other Stellenbosch collection sites. This suggests that the fynbos plants also store their seeds in serotinous structures. abundance of ophiostomatoid fungi within the Protea These species should also be surveyed for the presence infructescences is also influenced by other environmental of ophiostomatoid fungi, in order to obtain a more detailed parameters. Further studies, including more study sites and South African Journal of Botany 2005, 71(3&4): 307–311 311 environmental parameters, need to be undertaken in order to ADM, Walton DWH (eds) Invertebrate-Microbial Interactions. understand the ecology of these fungi and their Protea hosts. Cambridge University Press, New York, pp 133–153 While there is a decline in the number of infructescences Goldblatt P, Manning J (2000) Cape plants: a conspectus of the containing visible fruiting structures towards the onset of Cape flora of South Africa. Strelitzia 9: 7–35 spring, fungi in laboratory samples taken in June and Lee S, Groenewald JZ, Taylor JE, Roets F, Crous PW (2003) Rhyn- costomatoid fungi occurring on Proteaceae. Mycologia 95: 902–910 maintained at room temperature under dry conditions, Marais GJ (1996) Fungi Associated with Infructescences of Protea remained visible for a substantially longer period. This may Species with Special Reference to the Ophiostomales. 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