METSÄNTUTKIMUSLAITOKSEN TIEDONANTOJA 679, 1998 FINNISH FOREST RESEARCH INSTITUTE, RESEARCH PAPERS 679, 1998 Characterization and pathogenicity of Rhizoctonia spp. associated with nursery-grown conifer seedlings suffering from root dieback Ari M. Hietala VANTAAN TUTKIMUSKESKUS METSÄNTUTKIMUSLAITOKSEN TIEDONANTOJA 679, 1998 FINNISH FOREST RESEARCH INSTITUTE, RESEARCH PAPERS 679, 1998 Characterization and pathogenicity of spp. associated with nursery-grown conifer seedlings suffering from root dieback Ari M. Hietala Academic Dissertation in Forest Pathology To be presented, with the permission of the faculty of Agriculture and Forestry of the University of Helsinki, for public discussion in the lecture room of Viikin koetila (Koetilantie 5) on Ist of June 1998, at 12 o'clock noon. Ari Hietala 1998. Characterization and pathogenicity of Rhizoctonia spp. associated with nursery-grown conifer seedlings suffering from root dieback. Finnish Forest Research Institute. Research Papers 679, 1998. 41 + 44 p. Metsäntutkimuslaitoksen tiedonantoja 679, 1998. 41 + 44 s. ISBN 951 -40-1617-3 ISSN 0358-4283 Key words: Pinus sylvestris, Picea abies, Larix sibirica, uni- and binucleate Rhizoctonia, mode of infection, Rhizoctonia taxonomy Publisher: Finnish Forest Research Institute, accepted by Matti Kärkkäinen, Research Director, 23.4.1998 Authors Department of Plant Biology, P.O. Box 28 (Viikki 21), present FIN-00014 University of Helsinki. Finland, address: E-mail: ari.hietala@helsinki.fi Layout: Maija Räisänen Hakapaino Oy Helsinki 1998 3 Abstract A disease referred to as root dieback has caused considerable losses in nursery production in Finland and Norway. Both containerized and bare-root seedlings of Scots pine and Norway spruce are affected by the disease. Wilting of young shoots, drooping tops, retarded height growth and discoloration of needles are the shoot symptoms observed in diseased seedlings in nurseries. Previous studies indicated that the disease could be of complex nature involving both Rhizoctonia, particularly uninucleate isolates, and Pythium. The aim of this thesis was to further characterize Rhizoctonia species associated with diseased seedlings. In a case study, intensive isolations from roots of diseased Norway spruce seedlings showed that several Rhizoctonia species can be co-existing in the same root system. On the basis of cultural morphology, hyphal nuclear condition and anastomosis tests, the obtained isolates could be divided into 5 species: uninucleate Rhizoctonia sp. and four distinct binucleate Rhizoctonia species. One of these binucleate Rhizoctonia species anastomosed with the tester isolate of AG-I of genus Ceratobasidium. The remaining binucleate species could not be connected with any anastomosis group of this genus and further work applying various fruiting techniques is needed for their characterization. Under aseptic conditions binucleate Rhizoctonia infected only cortical cells in basal root regions; accordingly, the root growth of seedlings inoculated with binucleate Rhizoctonia was unaffected indicating that these species are not directly involved with the disease. Hyphal and cultural morphology and anastomosis tests confirmed that the Rhizoctonia isolates considered most pathogenic in preceding studies, and originating both from Finland and Norway, represent the same species which is characterized by almost uniform cultural morphology, uninucleate nuclear condition and possession of a single anastomosis group. In RAPD-PCR analysis, the isolates showed over 75-80 % similarity coefficients further confirming the cultural observations. A new method involving the use of aseptically grown host seedlings was developed for fruiting. The method worked with all the tested hosts: Scots pine, Norway spruce and Siberian larch. On the basis of basidial characteristics, the uninucleate Rhizoctonia sp. belongs to genus Ceratobasidium, which has previously been regarded as a genus having only binucleate anamorphs. The basidial characteristics of the uninucleate Rhizoctonia sp. fit into the morphological species concept of C. bicorne, but further comparison (nuclear condition, hyphal anastomosis) was not possible since this species, described parasitizing a moss in Denmark, has apparently never been cultured. Considering the different nuclear condition, uniformity in cultural conditions and 4 possession of a teleomorph for which no anamorph state has been described, there is only one conclusion to draw; a new Rhizoctonia species should be described for these uninucleate isolates. No differences were observed in the mode of infection on the three hosts. In the tip region, where protoxylem elements had not yet differentiated, proliferating hyphae formed aggregations on the root surface and within the intercellular spaces of the outer cortex. This gave rise to penetration hyphae that invaded the apical meristem and neighbouring vascular cylinder. Only actively growing roots were infected by the fungus; root exudates are suggested to induce the hyphal proliferation preceding the formation of penetration hyphae. Both under sterile and non-sterile conditions, the uninucleate Rhizoctonia sp. attacked particularly the tips of primary roots and long laterals. In pathogenicity tests, inoculation of pine and spruce seedlings with the uninucleate Rhizoctonia sp. resulted always in a considerably stunted and characteristic root system morphology whereas larch seedlings were less affected; possible differences in seasonal root growth patterns could account for this fact. It is possible that the broad shoot symptom list presented above may reflect differences in the causal pathogen. A systematic survey and comparison of the root system morphology and mycoflora of seedlings showing varying shoot symptoms is recommended to critically evaluate this proposal. 5 Contents List of publications 6 Acknowledgements 7 1 Introduction 9 1.1. Root dieback in Nordic countries 9 1.1.1. Norway 9 1.1.2. Sweden 10 1.1.3. Finland 10 1.2. Characterization of Rhizoctonia species 11 1.2.1. Morphological taxonomy: a historical review 11 1.2.2. Morphological taxonomy vs. modern concept 13 1.2.2.1. Genus Thanatephorus 13 1.2.2.2. Genus Ceratobasidium 16 1.2.2.3. Genus Waitea 18 1.2.2.4. Genus Tulasnella 18 1.2.2.5. Genus Sebacina 19 1.2.3. Rhizoctonia taxonomy: conclusions and future prospects 19 2 Aims of the study 21 3 Materials and methods 22 3.1. Characterization of Rhizoctonia isolates 22 3.2. Pathogenicity of Rhizoctonia isolates 23 4 Results and discussion 24 4.1. Binucleate Rhizoctonia: characteristics and role in the root dieback disease 24 4.2. Uninucleate Rhizoctonia isolates 26 4.2.1. Isolate characterization 26 4.2.2. Pathogenicity 29 4.2.3. Conclusions 31 4.3. Ongoing research 33 5 References 35 Appendix I-V 43 6 List of Publications This thesis is based on the following articles, which in the text will be referred to by their Roman numerals. I Hietala, A.M., Sen, R. & Lilja, A. 1994. Anamorphic and teleomorphic characteristics of a uninucleate Rhizoctonia sp. isolated from the roots of nursery grown conifer seedlings. Mycological Research 98:1044-1050. II Hietala, A.M. 1995. Uni- and binucleate Rhizoctonia spp. coexisting in the roots of Norway spruce seedlings suffering from root dieback. European Journal of Forest Pathology 25:136-144. 111 Lilja, A., Hietala, A.M. & Karjalainen, R. 1996. Identification of a uninucleate Rhizoctonia sp. by pathogenicity, hyphal anastomosis and RAPD analysis. Plant Pathology 45: 997-1006. IV Hietala, A.M. 1997. The mode of infection of a pathogenic uninucleate Rhizoctonia sp. in conifer seedling roots. Canadian Journal of Forest Research 27: 471-480. V Hietala, A.M. & Sen, R. 1996: Rhizoctonia associated with forest trees. In: B. Sneh, S. Jabaji-Hare, S. Neate & G. Dijst (eds.). Rhizoctonia species: taxonomy, molecular biology, ecology, pathology and disease control (pp. 351-358). Kluwer Academic Publishers, Dordrecht, Netherlands. Papers I-V are reprinted by permission from the publishers. 7 Acknowledgements This work was a direct continuation to my M.Sc. thesis; they were both carried out among forest pathologists at the Vantaa research centre of Finnish Forest Research Institute. I am grateful for these years: the friendships made, the helping hands offered and of course the facilities provided. Anna-Maija, Annikki, Arja, Brita, Eeva, Heikki, Jarkko, Juha, Kari, Kati, Kerttu, Maarit, Matti, Michael, Rauni, Ritva, Risto, Sakari, Sonja, Timo, Tuula - thank you. In addition, I need to thank prof. Kim von Weissenberg and the folks at the department of Plant Biology for the positive attitude in completing this process. I am most grateful to prof. Timo Kurkela for supervising me and writing those numerous recommendations to foundations. I am in debt to my co-authors, Robin Sen, Arja Lilja and Reijo Karjalainen, for the fruitful collaboration. Especially, I want to thank Robin Sen for teaching the "rookie" essential methods, philosophy and perseverance required in this marathon. The uncounted, educative and fun hours spent in Kari Korhonen's room will outlive. In addition, he read all the manuscripts with great care having a substantial impact when stumbling towards the final versions. In addition, I need to thank Kari Kammiovirta for his excellent assistance in the third paper. Before the reviewing process, the summary was critically read by Kim von Weissenberg, Arja Lilja and Timo Kurkela. The thesis was reviewed by prof. Everett Hansen from Oregon State University and by Dr. Antti Uotila from the University of Helsinki. I thank them all for the constructive and encouraging comments. Without the trust of foundations, the included studies could not have been started or completed. Lalli Laine, Veikko Hintikka, Kim von Weissenberg, Robin Sen and Timo Kurkela are thanked for encouragement and letters of recommendation. In chronological order, grants awarded by The Natural Resources Research Foundation of Finland, Kemira Research Foundation, The Niemi Foundation, Jenny and Antti Wihuri Fund, and The Leino Foundation are gratefully acknowledged. A grant by Suomen Kulttuurirahasto enabled me to fully concentrate on completing the thesis by writing the summary. In addition, I want to thank Arja and Sakari Lilja, Kari Korhonen, Kati Lipponen and prof. Eero Paavilainen for their support during those periods I had no outside funding. And last, but certainly not least, I thank my life companion Mari for her extreme patience and ever-lasting faith in me and the work I was obsessed with. 8 9 1 Introduction 1.1. Root dieback disease in Nordic countries 1.1.1. Norway The first serious root disease incidents on conifer seedlings in forest nurseries of the Nordic countries were observed in the 1970's in Norway (Sandvik 1985), where the disease had mainly affected containerized Norway spruce (Picea abies (L.) Karst.) seedlings. The term "root dieback" was adopted in association with shoot symptoms including wilting of young shoots, drooping tops, retarded height growth and discoloration of the foliage (Galaaen & Venn 1979). Root systems of diseased seedlings were partially or totally dead, characteristically very pigmented and had few, if any, non-pigmented root tips compared to healthy seedlings (Venn 1985). Losses have been considerable, resulting in a 4-5 % reduction in the production value of Norwegian forest nurseries, and the disease has been regarded as the most serious and persistent problem in production of containerized stock (Eterja and Austara 1990). Surveys for fungi associated with root dieback and subsequent pathogenicity experiments suggested that the disease could be of a complex nature involving several pathogens. In the first study (Galaaen and Venn 1979), Pythium sylvaticum Campbell & Hendrix was found to be prevalent on the roots of diseased 2-year-old Norway spruce seedlings, and based on a pathogenicity test using young germinants of Norway spruce under aseptic conditions, the fungus was suggested to be involved in the disease. Other associated fungi showing pathogenicity in that study included Botrytis cinerea Pers. ex Fr., Trichoderma viride Pers. ex Fr. and Fusarium avenaceum (Fr.) Sacc. Later Norwegian studies showed that Pythium spp. prevailed on the roots of diseased seedlings (Venn et al. 1986). In order to study the interaction of growth media, fungicides and pathogens associated with root dieback, Venn et al. (1986) set up extensive experiments where 10-week-old Norway spruce seedlings were inoculated with pathogens under nursery conditions. The pathogen candidates, a Pythium sp. and a Rhizoctonia sp., had been chosen on the basis of an unpublished experiment, where these two isolates had been highly pathogenic. Inoculations with the Rhizoctonia sp. resulted in considerably stunted shoot growth with symptoms of needle discolouration. Inoculations with the Pythium sp. showed a somewhat different effect: a small percentage of seedlings were killed whereas the surviving ones were only slightly stunted in shoot growth. Shoot wilting, drooping tops, 10 root-system morphology or the level of root damage were not reported for either pathogen (Venn et al. 1986). 1.1.2. Sweden Death of roots of nursery-grown containerized conifer seedlings showing stunted shoot growth and needle discoloration has also interested researchers in Sweden (Unestam et al. 1989; Unestam and Beyer-Ericson 1990). Isolated fungi included Pythium spp., Cylindrocarpon destructans (Zins.) Scholten, Alternaria alternata (Fr.) Keissler, Ulocladium atrum Preuss and Botrytis cinerea. Unestam et al. (1989) chose C. destructans as a model pathogen to study it's behaviour in roots of stressed Scots pine (Pinus sylvestris L.) seedlings; low light conditions, anaerobic root environment and fungicide treatment were found to predispose seedlings to invasion by this pathogen. 1.1.3. Finland In Finland, the first report of seedlings showing shoot wilting and drooping, needle discolouration and death of the root system is from a nursery in northern Finland (Jalkanen 1985). Later studies on a root disease of conifer seedlings in forest nurseries in southern and central Finland report a somewhat different list of symptoms: needle discolouration, stunted growth and partial or total root death (Lilja et al. 1992). In Finland, the term "root dieback" was adopted in association with the latter list of symptoms (Lilja et al. 1992). Both containerized and bare-rooted seedlings of Norway spruce and also Scots pine are being affected by the disease (Lilja et al. 1992). No estimates are available on the economical losses due to root damage of conifer seedlings in Finland, but based on the nursery inspectors' annual reports covering sampled nurseries, losses due to unspecified root rot can be very considerable in individual nurseries; in certain years in some nurseries, even 50 % of spruce or pine seedlings in inspected seedling lots have been discarded due to a disease classified as "root rot" (unpublished reports by the nursery inspectors of the Ministery of Agriculture and Forestry). Compared to Norway and Sweden, the Finnish surveys of fungi associated with root dieback disease have been more intensive. The relatively high isolation frequencies of a uninucleate Rhizoctonia sp., and of Pythium spp. and Phytophthora undulata (H.E. Petersen) M.W. Dick from diseased seedlings and their pathogenicity in tests suggested that these fungi are involved in the root dieback disease (Lilja et al. 1992). 11 1.2. Characterization of Rhizoctonia isolates Species of Rhizoctonia are übiquitous soil inhabiting fungi. Many species are associated with worldwide diseases on important crop plants, such as cereals, cotton, sugarbeet and potato. Besides pathogens, the genus includes also saprophytes and orchid-associated mycorrhizal fungi (see Sneh et al. 1991). A lot of taxonomic confusion has been related to the genus over the years. The purpose of the following review is to critically evaluate the past and present approaches taken in classification of these species. 1.2.1. Morphological taxonomy: a historical review The genus Rhizoctonia was established in 1815 by de Candolle to accommodate a non-sporulating root rotting fungus, Rhizoctonia crocorum (Pers.) DC. The basic characters of the genus set forth by de Candolle were quite liberal: formation of sclerotia with uniform texture and the association of hyphae with roots of living plants (the name Rhizoctonia originates from Greek and means "death of roots"). Like in other fungal genera, also in Rhizoctonia the taxonomy has been based on morphological characters and host species involved. In the absence of specific characteristics, nearly 100 species have been assigned to the genus (see Andersen & Stalpers 1994). Over the years, it became very clear that these Rhizoctonia species formed a heterogeneous group of fungi which were not closely related to each other. In addition, many of the species assigned to the genus did not even fit in the original frame set by de Candolle in 1815: some of these species had sclerotia which consisted of a rind and a medulla and some others were not associated with roots at all. It is perhaps a common misunderstanding among "non-Rhizoctonists" to regard Rhizoctonia species as fungi without a perfect state. Rhizoctonia species are not easily induced to fruit under laboratory conditions, and under natural conditions their inconspicuous fruitbodies are easily overlooked. During 1960'5, observations on the indigenous or laboratory induced teleomorphs revealed that species designated as Rhizoctonia contained both ascomycetes and basidiomycetes (e.g. Warcup & Talbot 1966). First observations on the nuclear condition of Rhizoctonia isolates were made over 70 years ago (Miiller 1924). Parmeter at al. (1967) were the first to compare the nuclear condition of vegetative cells and the perfect state of Rhizoctonia isolates. They found that isolates having a Thanatephorus cucumeris (Frank) Donk perfect state always had multinucleate vegetative cells whereas other isolates with teleomorph characteristics of the genus Ceratobasidium were binucleate. This discovery that Rhizoctonia isolates can be divided into two groups, multinucleate or binucleate, based on nuclear condition of vegetative 12 cells became a corner stone in laying Rhizoctonia characterization on a more solid ground. Parmeter et al. (1967) also pointed out that the vegetative characteristics of these isolates are often indistinguishable if nuclear condition is not determined. This work casts a dark shadow over species identifications in earlier reports. That work of Parmeter at al. (1967) resulted in a revision of taxonomic characteristics required for identification of the most studied Rhizoctonia species, R. solani Kiihn (Parmeter and Whitney 1970). Later, these characteristics were expanded to cover the whole genus (Ogoshi 1975). As a result, the genus Rhizoctonia was now proposed to include basidiomycetous imperfect fungi having the following characteristics: (a) branching near the distal septum of cells in young, vegetative hyphae; (b) formation of a septum in the branch near the point of origin; (c) constriction of the branch; (d) dolipore septum; (e) sclerotium not differentiated into rind and medulla; (f) absence of clamp connections, conidia and rhizomorphs. Since species of Ascomycota have simple pores in septa without a dolipore structure, these criteria restrict the genus to Basidiomycota. This has resulted in the exclusion of ascomycetous species from the genus (e.g. Sneh et al. 1991). Sneh et al. (1991) and Andersen and Stalpers (1994) have published checklists for Rhizoctonia including information on the species excluded from the genus, taxa that are considered to be doubtful due to poor descriptions, and synonomy. Of the morphological characters, the teleomorph is undoubtedly the most important single character, but due to the difficulty in fruiting these species under laboratory conditions, the isolate characterization is normally based on the vegetative stage. Vegetative characteristics such as mycelial colour, hyphal diameter, number of nuclei, length of cells, shape and size of monilioid cells and sclerotial size, have generally been used in the characterization of Rhizoctonia species. As pointed out by Andersen (1990), care is needed in the evaluation, as most of these characteristics vary considerably with temperature, light and composition of media. For example, hyphae have been reported to swell up to eight times their original diameter in a concentrated substrate (Burgeff 1936). The cell length, once thought to be of diagnostic value, was demonstrated in the measurements of Andersen (1990) to be of no value in characterizing taxa in Rhizoctonia. The dimensions of monilioid cells, criteria used by e.g. Saksena and Vaartaja (1961) when describing new species of Rhizoctonia, have been shown (Butler & Bracker 1970) to be of limited taxonomic value due to their high variability and dependence on the medium. On the basis of the structure of parenthesomes in dolipore septa, teleomorphic genera having Rhizoctonia anamorphs can be divided into two groups (Moore 1996). Determination of cell nuclear condition is a very useful first check when characterizing Rhizoctonia isolates; excluding this feature, R. solani and several other species belonging to genus Ceratobasidium are indistinguishable (Parmeter & Whitney 1970; Burbee et al. 1980). Lately, testing of anastomosis group and 13 the development of DNA based protocols have provided valuable tools for evaluation of traditional morphological taxonomy (see further text). 1.2.2. Morphological taxonomy vs. modern concept 1.2.2.1. Genus Thanatephorus This genus contains several teleomorph taxa, but an anamorph state is known only for Thanatephorus cucumeris (anamorph R. solani) (see Sneh et al. 1991). Most of the teleomorphs with an unknown anamorph state are associated with orchids (see Andersen & Rasmussen 1996). Considering its broad host range, übiquitous nature as a soil inhabitant and common association with some of the most important agricultural crops, it is only logical that of the species described as Rhizoctonia, R. solani has received the most attention. Again, the original description of the species (Kuhn 1858) was very vague causing a lot of confusion. The work of Duggar (1915) considerably clarified the taxonomy of R. solani, but the final breakthrough came only after Parmeter et al. (1967) compared the nuclear condition and teleomorphs of Rhizoctonia isolates. As a result, the taxonomic criteria required for the identification of R. solani were modified to the presently accepted concept (Parmeter and Whitney 1970). The specific criteria include the multinucleate condition of vegetative cells, production of brown pigments in culture, usually rapid growth rate and hyphae greater than 5 (im in diameter. The perfect state of R. solani, Thanatephorus cucumeris, belongs to family Ceratobasidiaceae (order Ceratobasidiales) of class Basidiomycetes (Hawksworth et al. 1995) and it has the following key characteristics: barrel-shaped to subcylindrical metabasidia, not urniform nor constricted about the middle and little wider than the supporting hyphae. The stout, usually straight sterigmata vary in number per basidium (4(2-7)) and in length but are usually at least as long as the metabasidia when mature. Germination of basidiospore by repetition is common (Talbot 1965). Studies using transmission electron microscopy (TEM) have shown that the parenthesomes in the dolipore septum are perforate in R. solani (Moore 1996). From the observations that this fungus causes numerous diseases on a very broad host range, its cultural variability on media, etc, R. solani has long been thought to contain many intraspecific groups and there have been many attempts to divide the species into rational groups, indeed. The first report on the occurrence of hyphal anastomosis reactions between Rhizoctonia isolates dates back several decades (Matsumoto et al. 1932). Since this pioneer work , several researchers have used hyphal anastomosis reactions as a basis for grouping Rhizoctonia solani isolates (e.g. Schultz 1936; Richter and Schneider 1953; Parmeter et al. 1969) but the meaning and usefulness of this kind of grouping remained unknown for a long period. Of the numerous approaches taken in the 14 classification of R. solani isolates, the method using hyphal anastomosis has by far been the most succesfiil. Isolates of Rhizoctonia are assigned to anastomosis groups (AGs) by pairing them with tester strains and observing the hyphal interactions. Several systems have been used for pairing: a) water agar in Petri dishes (e.g. Parmeter et al. 1969); b) cellophane placed on agar media (e.g. Parmeter et al. 1969); c) agar-coated slides or cover slips (e.g. Tu et al. 1969); d) bare objective slides (Kronland and Stanghellini 1988). The interactions of overlapping mycelia are examined under a light microscope. The hyphal interactions can be divided into four categories (Carling 1996). The reaction type often described as "perfect fusion" involves cytoplasmic fiision between the opposing hyphae and does not result in cell death. This is a typical reaction in a self-pairing. Excluding pairings between isolates originating from the same field, perfect fusion is a very rare event in nonself pairings. The term "imperfect fusion" or "killing reaction" refers to a reaction where the anastomosing cells and adjacent cells die after the cell wall fusion and no cytoplasmic connection is produced. Imperfect fusion is the typical interaction type between closely related isolates that belong to the same anastomosis group. In the third hyphal interaction type, the anastomosis reaction is characterized by apparent cell wall contact between the opposing hyphae but only occasionally one or both anastomosing cells and adjacent cells die. This type of anastomosis can be observed between distantly related isolates of the same anastomosis group. If the opposing hyphae just grow past each other without interaction, the isolates do not belong to the same anastomosis group (a negative reaction). Using this categorization, most of the isolates of R. solani can be easily placed into a certain AG. However, the existence of bridging isolates can sometimes make the reading of anastomosis tests difficult; the bridging isolates are isolates that are able to anastomose with isolates from more than one AG. In intergroup anastomosis, the interaction type is usually the same as observed between distantly related isolates of the same anastomosis group (Carling 1996). The genetic background of anastomosis behaviour in R. solani has not been studied, but a somatic incompatibility system has been offered as an explanation for the phenomenon (e.g. Anderson 1982). The genetic basis of somatic incompatibility is also poorly understood in other basidiomycetes, but like in Heterobasidion annosum (Fr.) Bref. (Hansen et al. 1993), the system could be controlled by several independent loci with probably multiple alleles at some loci (Adams 1996); for two heterokaryons to be somatically compatible, they would need to possess the same allele combination at these loci. Through meiosis and subsequent random mating of homokaryotic isolates, practically all heterokaryons, unless the nuclear pairs are closely related, will show differing allele combinations at these loci and are thus somatically incompatible. This would explain why the perfect fusion, if a somatic compatibility reaction, is so rare in anastomosis pairings between field isolates of Rhizoctonia. Following this reasoning, some researchers have used the anastomosis reactions as a tool 15 for mapping genotypes in a field and treated isolates producing a perfect fusion as a single clone (Ogoshi & Ui 1983; MacNish et al. 1993). Since related heterokaryons can show somatic compatibility, while still differing at other loci not regulating this feature, a term "vegetatively compatible population" has been proposed to describe R. solani isolates that produce the perfect fusion (MacNish & Carling 1995). Presently, Rhizoctonia solani is divided into 12 anastomosis groups (AG 1 - AGII, AG BI) (Carling 1996). No isolates of AGs 1, 4, 5, 7, 9, or 10 are known to bridge with isolates of any other AG. At least some isolates of the remaining six AGs bridge with certain AGs (Carling 1996). Different AGs cannot be separated from each other on the basis of vegetative morphology. Excluding AG 4, all the AGs show identical teleomorph characteristics. Isolates belonging to this group average three sterigmata instead of the common four observed in other AGs (Ogoshi 1976). For this reason, AG 4 isolates have often (e.g. Sneh et al. 1991) been classified under another teleomorph, namely Thanatephorus praticola (Kotila) Flentje. In the most recent taxonomic review, T. praticola has not been recognized as a separate taxon (Stalpers & Andersen 1996). Over the years, it became very clear that even the AG classification can be too general and most of the AGs have further been divided into subgroups, e.g. on the basis of colony morphology and pathogenicity anastomosis frequency with other members of the group, DNA homology and isozymes (see Sneh et al. 1991). Presently, there are still many geographic areas and host species where the information concerning the anastomosis grouping of associated isolates is lacking, but the available information indicates that the AGs and their subgroups show differences in host range or disease type. For example, isolates of AG 1 are mainly from the Leguminaceae and the Graminaceae, whereas most isolates of AG 3 are from the Solanaceae (Ogoshi 1987). As opposed to other groups, isolates of AGs 5, 6, 1,9, 10 and BI are considered to be weakly pathogenic or non-pathogenic (Sneh et al. 1991). The validity of anastomosis grouping and subgroup division has been examined in numerous studies using a variety of genetic and biochemical approaches. Supported by the results of studies based on DNA/DNA hybridization (Kuninaga & Yokosawa 1982ab; Kuninaga & Yokosawa 1983; Kuninaga & Yokosawa 1984ab; Kuninaga & Yokosawa 1985ab; Vilgalys 1988), restriction analysis of the ITS sequence (Liu & Sinclair 1993; Liu et al. 1993; Keijer et al. 1996) and isozymes (Liu et al. 1990; Liu & Sinclair 1992; MacNish & Sweetingham 1993), it has been concluded that not only are the AGs genetically different from each other but many of them can be broken into subgroups. Do the AGs or the subgroups represent independent biological species? The ultimate test would be a mating test and this would require homokaryotic isolates, but the difficulty in fruiting Rhizoctonia isolates under laboratory conditions has been a major obstacle. At least some AGs ( subgroup 1C of AG 1 and AG 4) have isolates that belong to outcrossing populations 16 with a mating system controlled by a single locus with multiple alleles (Puhalla & Carter 1976; Adams & Butler 1982). Initial studies suggest that the mating system of AG 8 is also bipolar (Yang et al. 1992). In addition, several AGs (AG 1 through AG 4) are known to contain isolates that are capable of homokaryotic fruiting (see Adams 1996). Because of the general lack of information about mating or mating relationships in most AGs, the taxonomic position of the AG grouping remains open. However, supported by the accumulating evidence, it is logical to conclude that the AGs and the subgroups represent, if not biological species, at least diverging evolutionary units (Anderson 1982). Therefore, the convenient label R. solani cannot be regarded as a description sufficient by itself in studies and reports concerning this fungus; to be able to make valid comparisons between results obtained under variyng conditions (environment, host, etc.), information about the anastomosis and genetic relationships of R. solani isolates should be included in all studies. For this purpose, standardized tester strains representing different AGs of R. solani are available in culture collections (e.g. American Type Culture Collection). 1.2.2.2. Genus Ceratobasidium Compared to R. solani, other Rhizoctonia species have received relatively little attention. Of these, the species belonging to genus Ceratobasidium are probably the most well-known. Morphological taxonomists consider this genus to be very closely related to the genus Thanatephorus (e.g. Stalpers & Andersen 1996). The teleomorph Ceratobasidium belongs to the family Ceratobasidiaceae (order Ceratobasidiales) of class Basidiomycetes (Hawksworth et al. 1995) with the major characteristics as follows: subglobose or obpyriform metabasidia which are 2 - 3 times the width of the supporting hyphae; sterigmata commonly four, sometimes fewer or more, about the same length as the metabasidia, sometimes forking; basidiospores germinate repetitively (Talbot 1965). Under TEM, the parenthesomes of the dolipore septum appear perforate like in R. solani (Moore 1996). In contrast to R. solani, the anamorphs of this genus have been regarded to be binucleate (Sneh et al. 1991). Excluding the nuclear condition, many of the anamorphs of Ceratobasidium are culturally indistinguishable from R. solani-, they produce brown hyphal and sclerotial pigments in culture, have relatively wide hyphae (5 (im) and are able to grow rapidly (Parmeter & Whitney 1970; Burbee et al. 1980). In addition, this genus contains species that, instead of some shade of brown, have hyaline, white or yellow-white colonies that are thus easily separated from R. solani. Fourteen different Ceratobasidium species are recognized in the most recent taxonomical review (Stalpers & Andersen 1996); apparently, most of these species have never been cultured since an anamorph state is known only for five of these Ceratobasidium species (see Sneh et al. 1991). Many of the teleomorphs for which an anamorphic state is unknown are associated with orchids (see Andersen & Rasmussen 1996). 17 The anastomosis group concept has also been applied to this genus. Ogoshi et al. (1979) divided Japanese isolates having a Ceratobasidium fruiting state into 17 AGs (AG-A - AG-O). In USA, Burbee et al. (1980) applied anastomosis testing and divided the local strains into 7 anastomosis groups (CAG-1 - CAG-7). Later, the relation of these groupings was examined and they were combined since five of the anastomosis groups corresponded to each other (Ogoshi et al. 1983). Presently the genus is divided into 21 AGs (AG-A - AG-S) (see Sneh et al. 1991). Like R. solani, these fungi are associated with various diseases on numerous hosts. In addition, some of the AGs are regarded as non-pathogenic (see e.g. Sneh et al. 1991). Most of the AGs of Ceratobasidium have an anamorph that has not been identified at species level and is designated only as Rhizoctonia sp. In addition, nine taxonomic species are included in the genus (e.g. R. endophytica Saks. & Vaar., R. callae Saks. & Vaar., R. fragariae Husain & McKeen and R. ramicola Weber & Roberts) (Sneh et al. 1991). All four example species belong to anastomosis group AG-A (Ogoshi et al. 1983). Likewise, most of the AGs have a teleomorph designated as Ceratobasidium sp.; only 8 AGs have a teleomorph identified at the species level. It is also noteworthy that five different AGs have a teleomorph identified as C. cornigerum (Bourdot) Rogers (see e.g. Sneh et al. 1991). Using RFLP analysis of PCR-amplified rDNA, Cubeta et al. (1991) were able to separate 13 of the 21 AGs into distinct groups which were consistent with the anastomosis grouping. Examining isolates of 12 AGs with isozymes, Damaj et al. (1993) identified five different groups which corresponded well with those rDNA groups. Parmeter et al. (1967) were able to fruit single spore isolates of a Ceratobasidium sp. but this is practically all that is known about the sexuality of Ceratobasidium species. The four species designated as members of AG-A show differing cultural morphology (e.g. Sneh et al. 1991) and merit a status of distinct taxonomical species on this basis. The question whether these species show genetic differences has not been addressed in any study using genetic markers. The facts that several AGs of Ceratobasidium are just designated as Rhizoctonia sp. with no specific morphological characteristics described and that some AGs share the same teleomorph (Ceratobasidium cornigerum) do not encourage the use of traditional morphological taxonomy in grouping binucleate Rhizoctonia isolates. Assessing the reports published on these fungi during the past 10-15 years, it seems clear that the traditional morphological characterization has been largely abandoned. Instead, after the determination of nuclear condition of isolates that morphologically fit into the genus concept, researchers go straight into anastomosis testing with tester strains of genera having a relevant nuclear condition. More information is clearly needed on the genetic relationships of the AGs of Ceratobasidium. In the mean time, encouraged by the observed genetic divergence among AGs of R. solani, it is also logical to treat the AGs as a basis for isolate characterization in this group when considering the morphological confusion related to these species. 18 1.2.2.3. Genus Waitea This genus contains two Rhizoctonia species, R. zeae Voorhees and R. oryzae Ryker & Gooch. These species are associated with diseases on hosts like corn, rice and cereals (Sneh et al. 1991). Like R. solani, these species have a fast growth rate and relatively wide hyphae with multinucleate cells. In contrast, their sclerotia are either reddish (R. zeae) or salmon coloured (R. oryzae) and often covered with a gelatinous layer; neither of these characteristics can be observed in R. solani (Stalpers & Andersen 1996). The teleomorph of both anamorphs is Waitea circinata Warcup & Talbot. According to Hawksworth et al. (1995), this genus belongs to family Botryobasidiaceae of order Tremellales, whereas Moore (1996) regards it as a member of family Ceratobasidiaceae of order Ceratobasidiales. The key characteristics of the perfect state are: metabasidia suburniform; sterigmata small and horn-like, four in number, about one-fifth to one-quarter the length of the metabasidium, and non-repetitive basidiospores (Talbot 1965). Under TEM, the parenthesomes of the dolipore septum appear perforate like in R. solani. Isolates representing these two anamorphs do not anastomose with each other. The two anastomosis groups recognized for the anamorphs of Waitea circinata are designated as WAG-Z (R. zeae isolates) and WAG-0 (R. oryzae isolates) (Sneh et al. 1991). 1.2.2.4. Genus Tulasnella The genus Tulasnella belongs to family Tulasnellaceae of order Tulasnellales (Hawksworth et al. 1995). This genus is very rich in species, containing over 30 teleomorph taxa. The main characteristics of the teleomorph genus are: subglobose, pyriform or sphaeropedunculate metabasidia, often twice as wide as the supporting hyphae; sterigmata subglobose to broadly ellipsoid at the base; spores germinate by repetition (Stalpers & Andersen 1996). In contrast to the previous genera, the parenthesomes in the dolipore septum are imperforate or pauciperforate in this genus (Moore 1996). The described teleomorphs have been mostly associated with fallen trunks and branches of forest trees (e.g. Roberts 1992; 1993; 1994). In addition, some teleomorphs have been connected with orchids ( e.g. Warcup & Talbot 1966; 1967; 1980). In contrast to the large number of teleomorphs, very few anamorphs have been described for these species. All the known anamorphs are associated with orchids (e.g. R. repens Bernard and R. anaticula Currah) (see e.g. Sneh et al. 1991). The most characteristic features of the vegetative state of these species include hyphae narrow in diameter (2 - 3.5 pm), binucleate cells and a very slow growth rate ( 3 mm/day) (Sneh et al. 1991). 19 1.2.2.5 Genus Sebacina The teleomorph genus Sebacina belongs to the family Exidiaceae of order Tremellales (Hawksworth et ai. 1995) and its most striking and characteristic single feature is the longitudinally septate metabasidia. Like in Tulasnella, the parenthesomes in the dolipore septum are imperforate or pauciperforate (Moore 1996). The anamorphs of Sebacina are very poorly known. A strain isolated from mycorrhizal rootlets of a conifer seedling and identified as R. globularis Saksena & Vaartaja (Saksena & Vaartaja 1960) (R. globularis is regarded as a nomen ambiguum, see Andersen & Stalpers 1994), has been fruited under laboratory conditions and it was identified as a Sebacina sp. (Warcup & Talbot 1966). Another verified case is orchid-associated (Sebacina vermifera Oberwinkler) (Warcup & Talbot 1967). The anamorphs of Sebacina resemble closely those of Tulasnella-, they are slow growers, possibly binucleate in vegetative state (the nuclear condition has been reported only for the R. globularis isolate) and there is overlap in the hyphal diameter between these two genera (Currah et al. 1990; Sneh et al. 1991; Warcup & Talbot 1967). 1.2.3. Rhizoctonia taxonomy: conclusions and future prospects Several basidiomycetous genera have anamorphs that fit into the present concept of Rhizoctonia but the difficulty in fruiting Rhizoctonia isolates under laboratory conditions has usually prevented the use of teleomorph characteristics in identification of the studied isolates. Therefore, it is understandable that the name Rhizoctonia has been almost exclusively used for these fungi, even if the perfect state was actually known. Since Rhizoctonia species do not form conidia, their vegetetative characteristics are quite limited in diagnostic features. As a result, many of the characteristics used in Rhizoctonia taxonomy overlap between different species. The adoption of anastomosis grouping as a routine practice in Rhizoctonia studies has provided valuable information for evaluation of the taxonomic species concept. The application of genetic markers in association with anastomosis tests has further shown that the species identification based on vegetative characteristics of these fungi can be too general. As a result, the traditional taxonomy based on vegetative characteristics has largely been displaced by anastomosis tests. For example, isolates are now being characterized as "binucleate Rhizoctonia belonging to AG-E of genus Ceratobasidium" (see e.g. Runion & Kelley 1993). This tendency is very acceptable. Presently, the anastomosis groupings of Rhizoctonia isolates are based almost exclusively on Japanese and American populations and there are still large geographic areas and unexamined hosts where anastomosis testing has not been applied. 20 Therefore, new AGs are likely to arise even within the most well-known Rhizoctonia species, R. solani. This is also probably the reason why Rhizoctonia isolates failing to anastomose with testers of putative genera are continuously being reported. The only way to sort out this situation is to put increased efforts in fruiting these presently unassignable isolates. Sneh et al. (1991) have presented a summary of methods used for fruiting Rhizoctonia isolates. The requirement for a dolipore septum in species acceptable as Rhizoctonia has created a nomenclatural problem; the type species of the genus, R. crocorum (teleomorph Helicobasidium (Desm.) has simple pores without the dolipore structure and would have to be excluded from the genus as proposed e.g. by Sneh et al. (1991). Unlike other teleomorphs of Rhizoctonia, the genus Helicobasidium does not belong to class Basidiomycetes but is included in class Ustomycetes (family Platygloeaceae of order Platygloeales) (Hawksworth et al. 1995). Taxonomically, it is not correct to exclude the type species from the genus and Moore (1987) has proposed that the name Rhizoctonia should be reserved for R. crocorum and related fungi which have septa with simple pores. He suggested that all the Rhizoctonia species possessing dolipore septa should be renamed according to their teleomorph genus. For example, anamorphs of genus Thanatephorus should be placed into genus Moniliopsis Ruhland. Because of the familiarity of plant pathologists with the name Rhizoctonia, this suggestion did not gain enthusiastic support. A compromise proposal has now been made to change the typification of Rhizoctonia by conservation and to adopt R. solani as the type species. This proposal has not yet been officially treated but its acceptance is anticipated (Stalpers and Andersen 1996). However, this proposal would restrict the name Rhizoctonia to anamorphs of the genus Thanatephorus and its acceptance will cause a nomenclatural chain reaction; the name Rhizoctonia in anamorphs of Ceratobasidium, Waitea, Tulasnella and Sebacina is proposed to be substituted with names Ceratorhiza, Chrysorhiza, Epulorhiza and Opadorhiza, respectively (Moore 1996). Considering the generic variability covered under the name Rhizoctonia, these nomenclatural proposals seem justified and hopefully will work to reduce the confusion that has surrounded these fungi ever since the genus Rhizoctonia was erected. On the other hand, what will those " Rhizoctonia " isolates unassignable in anastomosis tests and with an unknown perfect state be called? 21 2 Aims of the study Under the heading "Root dieback disease in Nordic countries", I have tried to briefly summarize the knowledge available on the disease at the time I finished off my M.Sc. thesis (Hietala 1992). One of the encouraging results of that work was that the fungus found most pathogenic in Norwegian experiments (Venn et al. 1986), a Rhizoctonia sp., turned out to represent the same species found common and pathogenic in Finnish studies (Lilja et al. 1992). Even at that time, it was obvious that this Rhizoctonia species, besides being pathogenic, also had some interesting taxonomic characteristics that together could justify the following studies: a) Characterization of Rhizoctonia species associated with root dieback b) Mode of infection of associated Rhizoctonia spp. The most comprehensive work on Rhizoctonia associated with trees was done almost 40 years ago (Saksena & Vaartaja 1960; 1961). Since that time the genus and species concepts of Rhizoctonia have evolved considerably. Therefore, substantial effort was required to determine the probable relationships of the isolates now under study. For this reason, the outcome of this literature review is included in the thesis (V). 22 3 Materials and methods 3.1. Characterization of Rhizoctonia isolates The Finnish isolates used in the study were either obtained from the collections of Aija Lilja (Finnish Forest Research institute (I, 111 & IV) or isolated during the study process (II). In both cases, the isolates were obtained by placing root segments of nursery-grown conifer seedlings, surface-sterilized with 0.5 % NaOCl, onto distilled water agar (Lilja et ai. 1992). The Norwegian reference isolates (I, III) were supplied from the collection of Kare Venn (Norwegian Forest Research Institute). To confirm that the isolates used in the studies (I, II) fit into the modern genus concept of Rhizoctonia, the isolates were examined for the criteria presented by Ogoshi (1975). Hyphal characteristics (growth pattern, hyphal diameter, septal structure and nuclear condition of cells) were examined after growth of the isolates on slides coated with low strength (1/8) potato dextrose agar (PDA). The septal condition was examined under phase contrast. Nuclei were stained with HCI-Giemsa according to the protocol of Wilson (1992) (1,11 & III). Colony characteristics were determined after 21 d growth on PDA at 24° C (I) or 21° C (II). The growth rates of the isolates on PDA were examined using a temperature series (7, 14, 18, 21, 24, 28, 31° C) (I) or at a single temperature (2 1° C) (II). To study hyphal anastomosis, isolates were paired on agar media by inoculating them at a distance of 2 cm. Pairings were incubated until the margins of opposing colonies overlapped; hyphal anastomosis reactions were examined under a light microscope (I, 11, III). The standardized tester strains of genus Ceratobasidium (AG-A to AG-S), obtained from Akira Ogoshi (Hokkaido University, Japan), were used to assess the grouping of the studied isolates (I, II). A new method was developed for fruiting isolates. The studied isolates were inoculated into a Petri-dish system containing three aseptically growing Scots pine (I, IV), Norway spruce (IV) or Siberian larch (IV) seedlings in distilled water. Petri-dishes were kept at room temperature with natural indirect lighting. Following the development of hymenia, samples were transferred to microscope slides for measurements of teleomorph characteristics. The genetic similarity of uninucleate Rhizoctonia isolates was examined with RAPD markers (III). DNA was isolated from PDA grown cultures using the protocol described by Lee and Taylor (1990). Three randomly constructed primers (primer 91298: GGA CGA TTC G; primer 91299: CGA TTC GGC G; 23 primer 91300: CGA GGT TCG C) were used for amplification under conditions slightly modified from those of Williams et al. (1990). Amplifications were repeated twice for each isolate and only reproducible bands were scored. The statistical analysis of RAPD data, similarity coefficients (DICE) and clustering analysis was based on the NTSYS program (Rohlf 1989). 3.2. Pathogenicity of Rhizoctonia isolates To study the pathogenicity and mode of infection of Rhizoctonia species isolated from nursery-grown Norway spruce seedlings displaying root dieback symptoms, 5-week-old aseptically growing Norway spruce seedlings were inoculated with isolates representing different anastomosis groups. Seedlings were incubated for four weeks and examined for root system morphology. The data were subjected to analysis of variance and Tukey's HSD test. To examine the infection characteristics, intact root systems were stained with trypan blue according to the protocol of Phillips and Hayman (1970) and examined under a microscope (II). To study the pathogenicity of a collection of uninucleate Rhizoctonia isolates originating from Finland and Norway, ten-week-old Scots pine and Norway spruce seedlings were inoculated with the fungal isolates and incubated for 8 weeks under greenhouse conditions and examined for root system characteristics. In another experiment, one- and two-year old Scots pine seedlings were inoculated with the uninucleate isolate nr. 264 and incubated for 7 months under greenhouse conditions before examination of root system characteristics. The data from both experiments were subjected to analysis of variance and Duncan's multiple range test (III). To further investigate pathogenicity of the uninucleate Rhizoctonia sp. and infection on observed hosts, 7-week-old seedlings of Scots pine, Norway spruce and Siberian larch were inoculated with three isolates originating from the respective hosts. After an incubation period of 5 weeks in a growth chamber, seedlings were transferred to a greenhouse and the experiment was harvested when the seedlings had reached the age of 7 months. Several root parameters were recorded for the seedlings both at the time of inoculation and harvesting. This data was subjected to analysis of variance and Tukey's HSD test. Fungal isolations and somatic compatibility tests were made to verify the presence of inoculated strains in the roots at the time of final harvesting. To screen the locality of infection, two entire root systems representing each treatment were stained according to the procedures described by Koske and Gemma (1989). In addition, all root systems harvested at the time of inoculation were similarly stained and examined under a microscope. Following this screening of infection and to examine the infection more closely, root pieces representing areas of interest were embedded in paraffin for microtome sectioning (IV). 24 4 Results and discussion 4.1. Binucleate Rhizoctonia : characteristics and role in the root dieback disease The case study (II) showed that several Rhizoctonia spp. can be obtained from the roots of diseased seedlings; binucleate Rhizoctonia isolates were frequently coexisting with uninucleate Rhizoctonia isolates in the same root system. Before this, binucleate Rhizoctonia had been frequently isolated also from diseased seedlings and from seedlings considered healthy (Lilja et ai. 1992) but not in association with uninucleate isolates. On the basis of cultural morphology (II), these binucleate Rhizoctonia isolates could be divided into four morphological groups. These groups could be easily distinguished from the uninucleate Rhizoctonia sp. on cultural characteristics (colony colour, zonation and sclerotial characteristics). In hyphal diameter and growth rate, on the other hand, the binucleate isolates were in most cases not substantially different from the uninucleate isolates. Therefore, care should be taken when isolating Rhizoctonia; to discover the whole range of species associated with the studied host, a preliminary screening can be quickly done just by inoculating the isolates on a characteristic medium, e.g. PDA. To avoid potential mixed cultures, all isolates should be ideally obtained as single cell or hyphal isolations. The morphological grouping corresponded well with the anastomosis behaviour of representative isolates; binucleate isolates anastomosed only with other isolates sharing the same morphological characters. A killing reaction was commonly observed in nonself pairings within a morphological group indicating that the isolates were closely related but genetically different. Based on similarity in morphological characteristics and anastomosis behaviour, these four binucleate groups can be regarded as four distinct species. Of the four anastomosis groups found for binucleate Rhizoctonia (II), only one could be connected with AGs described for the genus Ceratobasidium\ the isolates in question anastomosed with the culturally similar AG-I tester, producing the killing reaction, which indicates a close relationship (= common AG) between the Finnish isolates and the Japanese tester. The anamorph of AG-I is R. fragariae Husain & McKeen (Ogoshi et al. 1983). There are no other reports concerning AG-I or R. fragariae associated with conifer seedlings, as AG-I is normally associated with root rot in strawberry fields (Martin 1988). Since there was no indication of the teleomorph of the binucleate isolates 25 belonging to the remaining unassignable three groups, it may simply be that these isolates do not represent anamorphs of genus Ceratobasidium. For the moment, the other two teleomorph genera known for binucleate Rhizoctonia, Tulasnella and Sebacina, are culturally very poorly known and no AG grouping has been set up for them. On the other hand, the acknowledged AGs of genus Ceratobasidium are based on Japanese and American fungal populations. It is very likely that in the future many new AGs will be reported within this genus also. This will require increased efforts in fruiting the unassigned isolates, which are nowadays very frequently being reported due to the increased application of anastomosis testing in isolate characterization. Several methods have been described for fruiting Rhizoctonia isolates (see e.g. Sneh et al. 1991). When comparing the effect of co-existing uni- and binucleate strains isolated from the same Norway spruce seedlings, the seedlings inoculated with uninucleate isolates showed generally considerably poorer root growth than the seedlings inoculated with the binucleate strains (II). Unlike uninucleate Rhizoctonia isolates, binucleate isolates seemed to have no effect on the seedling growth parameters. Observations on the hyphal behaviour of uni- and binucleate isolates in the roots were in agreement with the root system morphology. Compared to seedlings inoculated with uninucleate isolates, roots inoculated with binucleate isolates had relatively few surface hyphae, except in the older parts of the main root. In this region, binucleate isolates commonly infected cortical cells with intensely stained monilioid hyphae filling the entire cell. This type of infection was rarely observed in the roots inoculated with uninucleate isolates. Unlike binucleate isolates, uninucleate isolates colonized particularly the root tips. Saksena and Vaartaja (1961) found that several Rhizoctonia species infected cortical cells of some conifer seedlings with monilioid cells in a poorly developed root system. However, the authors do not mention whether the root tips or the vascular cylinder were infected in these seedlings. In addition to conifers, the infection of root cortical cells with monilioid hyphae of Rhizoctonia has been reported in numerous studies on various hosts. The high intensity in staining of monilioid hyphae in contrast to undifferentiated hyphae reflects differences in the cell wall thickness. Sclerotia, the resting stage of Rhizoctonia fungi, are commonly composed of monilioid hyphae (see e.g. Sherwood 1970) and monilioid hyphae within host cells probably act as sclerotium-like dispersal and survival units for Rhizoctonia (e.g. Ferris et al. 1984). These results from the pathogenicity test (II) are in agreement with other studies made in Finland (Lilja et al. 1992; Lilja 1994). The fact that the AG concept has not been applied in all studies concerning binucleate Rhizoctonia does not allow direct comparisons between different studies. Anyway, it can be concluded that the studied binucleate isolates (II) are not directly involved in the actual root dieback disease. Concentration on uninucleate Rhizoctonia in later studies was thus justified. Whether the co-existed binucleate Rhizoctonia had already colonized roots when the seedlings were healthy or followed after 26 infection by uninucleate Rhizoctonia sp. remains an open question. Most of the studies on Rhizoctonia in relation to trees report these fungi as damping-off pathogens (V). All the performed Finnish studies are based on pathogenicity in older seedlings (Lilja et ai. 1992; Lilja 1994; II) and do not necessarily implicate non-pathogenicity in young seedlings at damping-off age. In addition, the binucleate isolates used in the third paper as reference isolates did show moderate pathogenicity, so the results obtained in the second study should not be generalized concerning all binucleate species or different genotypes. 4.2. Uninucleate Rhizoctonia isolates 4.2.1. Isolate characterization All six cultures examined (I) showed the general characteristics of Rhizoctonia. Basally constricted hyphae arose at acute angles behind the apices of the advancing hyphae and at right angles in the older hyphal regions. A dolipore septum was always observed near the point of origin of the branch. The sclerotia were not differentiated into a rind and medulla and no conidia, rhizomorphs or clamp connections were observed. Hyphal cells were predominantly uninucleate except for one isolate which contained a relatively high percentage (11 %) of binucleate tip and subapical cells. Culturally, the isolates were almost identical on PDA: distinctive, small, hazel-coloured regions within the otherwise buff-coloured surface hyphae gave cultures a spotted appearance. Surface-located and submerged, fulvous to umber coloured sclerotia were readily formed. Isolates fitting the above cultural description were confirmed to be predominantly uninucleate also in the later studies (11, III). In the massive literature on Rhizoctonia, there are very few papers reporting a uninucleate nuclear condition. Uninucleate Rhizoctonia isolates have been isolated from roots of winter wheat (Hall 1986). However, based on considerable differences in hyphal and colony morphology and in growth rate, these isolates are very unlikely to be related to the uninucleate isolates obtained from the roots of conifer seedlings. Burbee et al. (1980) found an isolate of R. quercus Cast, and an isolate of R. alpina Cast, to be uninucleate. Ogoshi et al. (1983) examined the same isolates and could not confirm that result; they found the R. alpina isolate to be binucleate and could not say whether R. quercus was binucleate or not. The isolates examined in those two studies originated in the 1930's (Castellani 1934). We have also examined these two isolates and they both contain sectors; certain areas have constantly uninucleate cells whereas others are invariably binucleate (Hietala & Sen, unpublished observations). This could explain the inconsistency between the earlier studies, resulting possibly from genetic changes occurred during the long storage in culture collections. The six studied isolates had very similar growth rates (I). Maximum growth 27 for two isolates was obtained at 21° C and for the others at 24° C. At these temperatures, the radial growth rates of the isolates were around 8 mm/24 h. In this respect, these isolates can be regarded as fast growing, not substantially differing from many isolates of R. solani (e.g. Mordue et al. 1989). The diameter of subapical hyphae of uninucleate isolates ranged between 5 and 8 |im (I, II), resembling R. solani also in this respect (e.g Parmeter & Whitney 1970). All the uninucleate isolates anastomosed with each other. In contrast to the perfect fusion observed in self pairings, the killing reaction was commonly formed in nonself pairings (I, 11, III). In nonself pairings, perfect fusions were observed only between three isolates, which all originated from the same nursery (III). Even in these pairing combinations the killing reaction was frequently observed. The genetics of anastomosis reaction types are not known in these fungi but the killing reaction is usually regarded as a somatic incompatibility reaction, whereas perfect fusion is interpreted as a somatic compatibility reaction (Anderson 1982). Perfect fusions have been very rare in nonself pairings in other studies except between some isolates originating from a common field; in these cases, perfect fusion has been interpreted as a sign of clonality (Ogoshi & Ui 1983; MacNish et al. 1993). Therefore, it seems logical to interpret the killing reaction commonly observed in nonself pairings of the uninucleate isolates as a sign of genetic difference. No positive anastomosis reactions were observed when the isolates were paired against the R. alpina and R. quercus isolates indicating that the species are not related (I). In the RAPD analysis (III), the uninucleate Rhizoctonia isolates showed high similarity within the group, over 75-80 % similarity coefficients in a dendrogram analysis and differed drastically from the binucleate reference isolates used in the study. The Norwegian isolates included did not group together closely, but showed more similarity with certain Finnish isolates. In addition, the Finnish uninucleate isolates did not group together according to their geographic origin. Using the novel method employing Scots pine seedlings (I), all isolates excluding one (isolate 256) could be fruited. In a further experiment (IV), representative isolates (including isolate 256) fruited in the presence of all the hosts observed for this uninucleate Rhizoctonia. No fruiting was observed in either experiment when the seedlings were absent from the system. On the basis of basidial characteristics (I), these isolates can be placed into genus Ceratobasidium (Talbot 1965). The ratio between the widths of metabasidia and their supporting hyphae was generally over two, which is a major feature defining the genus. The observed development of basidia on basal hyphae or on short side branches and the repetitive germination of basidiospores also connect the uninucleate isolates to this teleomorph genus. In the anastomosis testing, the chosen two isolates did not anastomose with testers of any of the 21 AGs described for the genus. Compared to other members of this genus, the nuclear condition of these isolates is very unusual; the genus Ceratobasidium has been regarded to contain only binucleate Rhizoctonia (e.g. Parmeter & Whitney 1970; 28 Ogoshi et ai. 1983; Sneh et ai. 1991). The teleomorph characteristics of the uninucleate isolates resemble very closely those of C. bicorne Erikss. & Ryvarden, described from Danish field material, parasitic on the moss Polytrichum attenuatum (Brid.) (Eriksson & Ryvarden 1973). There are very few observations on C. bicorne. Besides the sample on which the species description is based, there are only two additional specimens of the teleomorph; one was found on a Polytrichum moss and the other one on bark of living Norway spruce in Bayern, Germany (Luschka 1993). The characteristics of basidia and basidiospores of C. bicorne are easily distinguished from other Ceratobasidium species (see e.g. Stalpers & Andersen 1996). The teleomorph characteristics of the uninucleate isolates fit well into the taxonomic (= morphological) species concept of C. bicorne. Further comparison (vegetative morphology and cytomorphology, anastomosis interaction) between the uninucleate Rhizoctonia isolates and the C. bicorne observed under natural conditions was not possible, since C. bicorne has apparently never been cultured. Based on vegetative and teleomorph characteristics, it can be concluded that the uninucleate Rhizoctonia isolates represent a single species. This species seems to be very common in Finnish forest nurseries around the country (Lilja 1994). From Norway, the other country reporting root dieback of Norway spruce, there are no data on the occurrence of the species. The aberrant nuclear condition makes this Rhizoctonia species very easy to distinguish from other Rhizoctonia species and, it raises the question whether the present root dieback disease is the first time the vegetative stage has been observed. The literature on Rhizoctonia associated with trees (V) may provide some answer. First, it is evident that the genus Rhizoctonia has received relatively little attention among pathologists and mycologists dealing with trees. It is also true that even today papers are being published without sufficient characterization of the isolates in question. For example, there are many recent papers on tree diseases, where the pathogen has been identified as R. solani without presenting any criteria for this judgement. As shown in several studies (e.g. Parmeter et al. 1967; Burbee et al. 1980), unless nuclear or teleomorph condition is known, R. solani and many binucleate Rhizoctonia are practically indistinguishable. The vegetatitive characteristics of the present uninucleate Rhizoctonia sp. (production of brown pigments, relatively wide hyphae and fast growth rate) are also similar to those of R. solani (see e.g. Ogoshi 1975) and there is thus clearly room for error. Therefore, one can only present worthless speculations about whether or not the uninucleate Rhizoctonia sp. has been observed before or is associated with roots of conifer seedlings only in Finland and Norway. 29 4.2.2. Pathogenicity Under non-sterile conditions, inoculations with uninucleate strains considerably reduced the root growth of both Scots pine and Norway spruce seedlings, but did not usually result in death of seedlings (III). In both Scots pine and Norway spruce significant reductions (Duncan's multiple range test, p = 0.05) in parameters related to main root length, lateral root length and root dry weight were observed for practically for all the seventeen uninucleate isolates tested. Clear reduction was also shown in the shoot dry weight of both hosts, but the difference to the control seedlings was statistically significant only in Norway spruce seedlings. There seemed to be no connection between the original host and isolate pathogenicity; all the isolates were approximately equally pathogenic in both hosts, when comparing the individual root parameter values of seedlings inoculated with each isolate against the mean value for all isolates within each host. Similar growth reduction patterns were also observed in the other experiment (III), where one- and two-year-old Scots pine seedlings were inoculated with an isolate originating from this host. None of the seedlings were killed during this experiment. The root staining methods developed for studying infection by VA mycorrhizal fungi proved to be very useful, not only for locating Rhizoctonia on the roots, but also for studying the mode of infection. The characteristic branching pattern of Rhizoctonia hyphae makes them easy to trace in a non-sterile system. No substantial difference was observed in the resolution of the two protocols (11, Phillips & Hayman 1970; IV, Koske & Gemma 1989), but the latter protocol is preferrable since it does not involve the use of toxic lactophenol. Under aseptic conditions, Norway spruce seedlings inoculated with uninucleate Rhizoctonia isolates displayed root systems with significantly stunted main and lateral roots (Tukey's HSD, p = 0.05). Staining of roots without sectioning showed hyphal proliferation at the root tips and suggested that the fungus penetrated the vascular cylinder via the root tips (II). The observations of the second study (II) are in agreement with the results obtained under non-sterile conditions for all three hosts (IV). The uninucleate Rhizoctonia isolates reduced the lateral and main root growth of all host species (Fig. 1) although the reduction in Siberian larch was not always significant (Tukey's HSD, p = 0.01). No host specificity was observed; seedlings inoculated with isolates originating from different hosts did not show substantially differing root or shoot parameters. For the three hosts, the only statistically significant reduction in shoot parameters (length and dry weight) was observed in Scots pine. The shoots of inoculated Norway spruce seedlings were clearly although statistically insignificantly reduced in growth whereas inoculated Siberian larch seedlings showed equal shoot growth to the uninoculated control seedlings. Combining microtome sectioning in the root analysis (IV) confirmed the observations made on the stained unsectioned roots (11, IV); in the root tips, the 30 Fig. 1. Root system characteristics of Norway spruce inoculated with the uninucleate Rhizoctonia sp. (IV). 1A: An uninoculated control seedling (left) and a seedling inoculated (right) at the age of 7 weeks. Seedlings were harvested at the age of 7 months. 1B: A close-up of the inoculated seedling in Fig. IA. The root tips of long roots are commonly missing in infected seedlings and the remaining root tips are heavily pigmented and arrested in growth giving the root system a stunted appearance. uninucleate Rhizoctonia sp. penetrated into the vascular cylinder. The prepenetration stage was characterized by formation of hyphal aggregates on the root surface giving rise to penetration hyphae. Rhizoctonia solani has been shown to form similar hyphal aggregations, termed infection cushions, on several plants (e.g. Dodman & Flentje 1970). The now observed aggregations on conifer roots were commonly hundreds of (am long and there seemed to be a close connection between the formation of penetration hyphae and the state of root growth. No penetration hyphae were observed on such roots, which on the basis of a present metacutization layer, were regarded to be in dormancy. In addition, penetration hyphae were seldom formed in actively growing roots in the region were first protoxylem elements had already differentiated. Considerable hyphal proliferation was observed when isolates were grown under membrane-isolated host roots. From these observations, it is concluded that the uninucleate Rhizoctonia sp. infects actively growing roots and root exudates probably provide energy for the luxurious hyphal proliferation preceding the formation of penetration hyphae. Components of root exudates of some conifer seedlings have been shown to stimulate hyphal growth of Rhizoctonia (Agnihotri & Vaartaja 1969) and sporangium germination of Pythium (Agnihotri & Vaartaja 1967). Moreover, there is considerable evidence from other hosts that plant exudates are essential for the formation of infection cushions of R. solani (e.g. Dodman & Flentje 1970; Armentrout et al. 1987). 31 At the time of inoculation, both Norway spruce and Siberian larch had a high percentage of dormant roots. Unfortunately, possible differences in seasonal root growth pattern between different hosts were not examined by sequential harvesting. Possible differences in growth patterns could partially account for the fact that larch seedlings were less affected by the pathogen, since no differences were observed in the actual infection sites in different hosts. Presently, there is very limited data about seasonal growth patterns of fine roots in tree seedlings (Wilcox 1954: Abies procera Rehd.; Wilcox 1968: Pinus resinosa Ait.; Johnson-Flanagan & Owens 1985: Picea glauca (Moench) Voss) and further research in this area should be interesting from several angles, not only from a pathologist's point of view. 4.2.3. Conclusions It would be unwise to assume that the uninucleate Rhizoctonia isolates and the C. bicorne teleomorphs found in natural conditions share the same vegetative characteristics (e.g. uninucleate nuclear condition), belong to a common anastomosis group and further, represent a single biological species. This was the reasoning behind the rather cautious taxonomic conclusion in paper I: "in conclusion, this uninucleate fungus does fit into Ceratobasidium but because of the unusual nuclear condition we would prefer to describe it as a uninucleate Rhizoctonia sp. having a Ceratobasidium fruiting stage". Ideally, a taxonomic species should represent a single biological species. However, there is ample evidence that this would not be the case with many Rhizoctonia related taxa. Thanatephorus cucumeris, Ceratobasidium cornigerum and Waitea circinata are probably species complexes, considering their subdivision into anastomosis groups. In this light, the taxonomic conclusion of paper I has elements of an understatement. There are no reasons why these uninucleate Rhizoctonia isolates could not be treated as anamorphs of a morphologically identical teleomorph, C. bicorne. Future will tell whether this uninucleate Rhizoctonia sp. and C. bicorne as described by Eriksson and Ryvarden (1973) are truly conspecific. Hyphal anastomosis data would indicate that the uninucleate isolates represent distinct but closely related genotypes and that the species includes a single anastomosis group. RAPD analysis does not support any further division among these isolates. It appears that the dual nomenclature for these fungi (anamorph vs. teleomorph) will be maintained in the future because of the difficulties in fruiting Rhizoctonia isolates. Considering the uninucleate nuclear condition, uniformity in cultural characteristics, high genetic similarity and possession of a teleomorph for which no anamorph state has been described, there is only one conclusion to draw; a new Rhizoctonia (or Ceratorhiza) species could and should be described for these uninucleate isolates. In the mean time, these uninucleate isolates can be referred to with the description "uninucleate Rhizoctonia sp. (or 32 Ceratorhiza sp.) having a Ceratobasidium bicorne perfect state". Nuclear condition has been treated as a key character in distinguishing anamorphs of the related two teleomorph genera, Ceratobasidium and Thanatephorus . Before the present study, only binucleate Rhizoctonia spp. have been known to possess a Ceratobasidium fruiting stage. Is there now a need for a taxonomic rearrangement due to the aberrant nuclear condition? The fact is that presently the nuclear condition is still unknown for several Ceratobasidium species and there is no quarantee that they will show the familiar binucleate nuclear condition. In addition, practically nothing is known about the sexuality of binucleate Rhizoctonia and the key question is: are they heterokaryotic and thus heterothallic? If they are, uninucleate isolates could represent homothallic clones or species. Therefore, an unusual nuclear condition, in itself, is most certainly not a sound basis for any taxonomic rearrangements. Phylogenetic data would be desperately needed to address this kind of questions. This approach would also be most welcomed in order to critically evaluate the validity of several Rhizoctonia taxa (e.g. anamorphs that belong to AG-A of genus Ceratobasidium). Lilja (1994) has first presented a hypothesis that root dieback may be a disease of successive infections. Primary infection by uninucleate Rhizoctonia results in a high moisture content in the growth substrate and wet conditions favour zoosporic fungi like Pythium. This is a sound conclusion, and it is striking that after the work of Venn at al. (1986) Norwegian studies on root dieback disease have concentrated on Pythium regarding these fungi as major pathogens and practically ignoring Rhizoctonia (Borja 1995). Seedlings inoculated with uninucleate Rhizoctonia sp. appear to show very characteristic root system morphology (II; IV). Unfortunately, available information on root system morphology of diseased nursery seedlings (Galaaen & Venn 1979; Venn 1985; Lilja et al. 1992; II) and seedlings inoculated with different pathogens (Venn et al. 1986; Lilja et al. 1992; Lilja 1994) lacks sufficient details for definitive conclusions. However, I would claim that in pathogenicity tests (11, 111, IV), the resulting root system morphology corresponds well with the one observed in those seedlings, from which I have isolated this species. Wilting of young shoots, hanging tops, discolouration of needles, retarded height growth and partial or total death of root systems are the symptoms related to the root dieback disease in Norway (Venn et al. 1986). In Finland, studies have concentrated on seedlings showing needle discolouration, stunted growth and partial or total root death (Lilja et al. 1992; Lilja 1994; II) but, as reported by Jalkanen (1985), we do have seedlings also showing wilting and hanging tops. Although the present study was focused on stunted nursery seedlings showing no shoot wilting or hanging tops, it raises a question whether the broad symptom list presented by Venn et al. (1986) actually reflects differences in the causative agent. I have occasionally inspected nursery seedlings showing shoot wilting and top hanging; in these particular seedlings, the root system has been 33 totally dead but structurally normal (i.e. lateral roots and main roots have not appeared stunted in growth). In addition, these roots invariably hosted Pythium spp. but no Rhizoctonia. A systematic survey and comparison of the root system characteristics and mycoflora of seedlings showing either wilting and hanging tops or shoot stunting would be needed to critically evaluate this proposal. This will require increased collaboration between nursery inspectors and pathologists, as nursery managers often tend to keep a low profile on their disease incidences. In Norway, root dieback losses are reported to have decreased following a) removal of old sand beds, b) implementation of container sanitation between crops and c) development of appropriate fungicide and irrigation regimes (e.g. Borja & Austara 1990). The results of Venn et al. (1986) implicated supporting sand beds act as an inoculum source for Rhizoctonia associated with root dieback. Most Rhizoctonia spp. probably survive in soil between annual crops particularly as sclerotia formed in colonized plant debris This is evidently the case with the uninucleate Rhizoctonia sp. also, which is characterized by abundant sclerotial production under cultural conditions (I). Several edaphic factors may influence survival of sclerotia; e.g. survival of R. solani in moist soil is considered to be lower than in dry soil. Under dry conditions, the sclerotia have remained viable several years (see Sherwood 1970). Therefore, the measures a) and b) can be recommended as a control procedure in a Rhizoctonia disease case also. However, ecologically (e.g. moisture requirement) and structurally (e.g. cell wall composition choice of fungicides) Rhizoctonia and Pythium show striking differences and will require different approaches. Sorting out the raised etiological questions will give us a more complete picture of the root dieback disease and would allow consideration of sensible target measures to control the primary pathogen in question in each particular case. 4.3. Ongoing research Development of DNA-based methods allow testing the crucial question whether the now studied uninucleate isolates and C. bicorne are truly conspecific. Preliminary results based on ITS-PCR (primers ITSI-F and ITS4-B, Gardes & Bruns 1993) combined with RFLP indicate at least a close relationship between them; the restriction patterns of the ITS-region of the uninucleate isolates and a C. bicorne herbarium specimen (on Polytrichum moss found in Luschka's (1993) survey in a forest in Bayern, Germany) are the same and differ from binucleate Rhizoctonia isolates and tested herbarium samples of other Ceratobasidium species found in Finland (Hietala, Sen & Hantula, unpublished results). When sibling single-spore isolates of this uninucleate Rhizoctonia species are paired with each other, no mating reactions (e.g. tuft formation) have been observed and a killing reaction is commonly formed in a majority of pairings. 34 Single-spore isolates can also be fruited and the killing reaction is common in sib-pairings of the second generation, too (Hietala, Hantula, Korhonen & Sen, unpublished results). Therefore, it is clear that this fungus is homothallic and being uninucleate, the observed somatic incompatibility (= killing reaction) in pairings between sibling single-spore isolates makes the species a very interesting model organism for future research. We started this "freetime project" already back in 1994 and it is not due to lack of efforts or tools that we can presently provide no explanation for this highly interesting phenomenon. The high genetic homogeneity of the uninucleate Rhizoctonia sp. (Ill) could implicate a narrow source population, resulting e.g. from seedling exchange between different nurseries. Analysing mitochondrial genes could sort out this question. Field isolates derived from different nurseries invariably produce the killing reaction when paired. In addition, several genotypes, based on observed killing reaction in pairings, can be found in a single nursery (III). These observations do not exclude the possibility of a common origin since killing reactions are commonly formed in pairings between sibling single-spore isolates. However, they would implicate that the fungus is fruiting in the nurseries or nearby. Under natural conditions, fruit bodies of Rhizoctonia have been recorded on a wide variety of hosts, including some broadleaved trees, and on surrounding soils. In fields, the fruit bodies of Rhizoctonia usually develop at relatively high temperature ( 20° C) and moisture conditions ( 90 RH) at the lower side of infected leaves but also on the surface of the lower stem (see e.g. Naito 1996). Compared to hyphal growth, airborne basidiospores can disseminate rapidly over long distances which could be of importance in the development of disease epidemics. It is not uncommon to isolate uni- and binucleate Rhizoctonia spp. from the lower stems of seedlings suffering from root dieback (Hietala, unpublished observations) and this possible fruiting place is worth further look. In addition, Polytrichum mosses are quite common in Finnish forests and also in our forest nurseries and an investigation of their mycoflora will form another logical investigation line. On the basis of anastomosis groups, most of the binucleate isolates studied in the second paper could not be assigned to known anastomosis groups of genus Ceratobasidium. Further work using various fruiting techniques is needed to place these isolates and to see whether Ceratobasidium testers were actually valid. The method developed for uninucleate Rhizoctonia sp. (I) does not seem to work for binucleate Rhizoctonia associated with root dieback of seedlings (Hietala, unpublished results). In pathogenicity tests of Lilja (1994), some binucleate Rhizoctonia seemed to promote seedling growth. This is quite interesting, since on the basis of anastomosis affinity and common ITS-RFLP patterns, certain binucleate isolates derived from roots of nursery-grown conifer seedlings would seem to be related to orchid-associated Rhizoctonia (Hietala, Zelmer & Sen, unpublished results). Orchids represent an example of hosts for which the anastomosis groups of associated Rhizoctonia have not been tested. 35 5 References Adams, G.C. 1996. Genetics of Rhizoctonia species. In: B. Sneh, S. Jabaji-Hare, S. Neate & G. Dijst (eds.). 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Perfect states of some Rhizoctonias. Trans. Br. Mycol. Soc. 49: 427-435. Warcup, J.H. & Talbot, P.H.B. 1967. Perfect states of Rhizoctonias associated with orchids. New Phytol. 66: 631-641. Warcup, J.H. & Talbot, P.H.B. 1980. Perfect states of Rhizoctonias associated with orchids. 111. New Phytol. 86: 267-272. Wilcox, H. 1954. Primary organisation of active and dormant roots of noble fir, Abies procera. Am. J. Bot. 41: 812-821. Wilcox, H. 1968. Morphological studies of the root of red pine, Pinus resinosa. Am. J. Bot. 55: 247-254. Williams, J.G.K., Kubelik, A.R., Livak, K.J., Rafalski, J.A. & Tingey, S.V. 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acid Research 18: 7213-7218. Wilson, A.D. 1992. A versatile giemsa protocol for permanent nuclear staining of fungi. Mycologia 84: 585-588. Yang, H.A., Tommerup, 1.C., Sivasithamparam, K. & O'Brien, P.A. 1992. Heterokaryon formation with homokaryons derived from protoplasts of Rhizoctonia solani anastomosis group eight. Exp. Mycol. 16: 268-278. I 1044 Mycol. Res. 98 (9): 1044-1050 (1994) Printed in Great Britain Anamorphic and teleomorphic characteristics of a uninucleate Rhizoctonia sp. isolated from the roots of nursery grown conifer seedlings ARI M. HIETALA, ROBIN SEN* AND ARJA LILJA Department of Forest Ecology, Finnish Forest Research Institute, P.O. Box 18, FIN-01301 Vantaa, Finland Vegetative, anastomosis, fruiting and basidial characteristics were analysed in one Norwegian and five Finnish Rhizoctonia isolates from the roots of various nursery grown conifer seedlings. The isolates displayed common hyphal and colony morphology that also confirmed their designation as a Rhizoctonia sp. Hyphal cells were predominantly uninucleate except for one isolate which contained a relatively high number (11%) of binucleate tip and subapical cells. All isolates had similar temperature dependent growth rates and formed a single anastomosis group within which killing reactions were detected in opposing fusion cells of all non self pairings. Fruiting was induced in all but one Finnish isolate using a novel method involving axenic liquid culture of the fungi in the presence of Scots pine (Pinus sylveslris) seedlings. Basidial and basidiospore dimensions indicate that the isolates represent a single Ceratobasidium sp. although two selected isolates showed no hyphal anastomosis reactions with binucleate testers of the different Ceratobasidium anastomosis groups (AG-A to AG-S). The identified teleomorph closely resembles C. bicorne but further confirmation was not possible because this fungus is only available as herbarium material. Rhizoctonia DC. was established in 1815 by de Candolle to accommodate a non-sporulating root rotting fungus, Rhizoc tonia crocorum DC.: Fr. However, the subsequent description of numerous heterogenic Rhizoctonia species prompted a major revision of the taxonomic criteria required for the identification of R. solani Kiihn that were later expanded to cover the whole genus (Parmeter & Whitney, 1970; Ogoshi, 1975). Accepted vegetative features included the requirements of hyphae with dolipore septa, basally constricted branching near the distal septum of cells, absence of clamp connections, conidia and rhizomorphs and sclerotia with undifferentiated structure. On the basis of the known teleomorphs the genus is limited to the sub-division Basidiomycotina; class Hymenomycetes (sub-class Holobasidiomycetidae or Phragmobasidiomycetidae) (Sneh, Burpee & Ogoshi, 1991). The four main genera represented are Thanatephorus Donk (includes the teleomorph of e.g. R. solani), Waitea Warcup & P. H. B. Talbot (e.g. R. zeae Voorhees), Tulasnella J. Schröt. (e.g. R. repens Bernard) and Ceratohasidium Rogers (e.g. R. endophytica var. endophytica H. K. Saksena & Vaartaja and R. cerealis E. P. Hoeven). A major taxonomic feature of the anamorphs that separates the former and latter two genera is the respective presence of multinucleate and binucleate cells in young vegetative hyphae (Sneh et al., 1991). The multinucleate R. solani (T. cucumeris (A. B. Frank) Donk and T. praticola (Kotila) Flentje and binucleate Rhizoctonia spp. ( Ceratohasidium spp.) have been respectively further sub-divided into 11 and 21 anastomosis * Present address: Department of General Microbiology, P.O. Box 41 (Mannerheimintie 172), FIN-00014 University of Helsinki, Finland. groups (AG) based on affinity and fusion of interacting hyphae of paired cultures (Sneh et al, 1991 and references therein). Isolates of two species, R. quercus Cast, and R. alpina Cast., were found to contain uninucleate hyphae (Burpee et al, 1980) but this could not be confirmed by Ogoshi et al (1983) who regarded the latter fungus to be binucleate. The anamorph of an authenticated uninucleate Rhizoctonia sp. from the roots of winter wheat has been described (Hall, 1986). Many Rhizoctonia species are economically important plant pathogens in agriculture and thus receive considerable attention but less is known about these fungi and their effect on forestry production, e.g. in forest tree nurseries. It has been known for a long time that damping-off in nursery conifer seedlings can be caused by R. solani (Vaartaja & Cram, 1956; Saksena & Vaartaja, 1961). More recently in Georgia, U.S.A., Huang & Kuhlman (1990) showed that R. solani and a binucleate Rhizoctonia sp., representing anastomosis groups AG-4 and CAG-3, respectively, were able to induce damping off symptoms in nursery Slash pine (Pinus elliottii Engelm. var. elliottii ) seedlings. A binucleate Rhizoctonia sp. (CAG-3) was also identified causing seedling blight of longleaf pine (P. palustris Mill.) in Florida (English, Ploetz & Barnard, 1986). Ten different Rhizoctonia species causing root rot in pine (P. sylvestris L. and P. resinosa Ait.) seedlings in Canadian nurseries included R. callae Cast., R. globularis H. K. Saksena & Vaartaja and R. endophytica var. endophytica (Saksena & Vaartaja, 1961). All have since been confirmed to be binucleate Rhizoctonia species and the latter is an anamorph of Ceratohasidium comigerum (Bourdot) Rogers (Sneh et al., 1991). In the Nordic countries, a binucleate Rhizoctonia sp. has been identified killing the needles of Norway spruce (Picea A. M. Hietala, R. Sen and A. Lilja 1045 Tabic 1. Rhizoctonia isolates from the roots of nursery grown conifer seedlings Abies (L.) Karst.) seedlings (Roll-Hansen & Roll-Hansen, 1968) and, more recently, an uncharacterized Rhizoctonia sp. was also found to cause root dieback in seedlings of the same tree species in Norwegian nurseries (Venn, Sandvik & Langerud, 1986). A nursery survey of fungi present in the roots of both bare-rooted and containerized grown conifer (P. sylvestris and P. Abies) seedlings in Finland included a uninucleate Rhizoctonia like fungus that was found to be an aggressive root pathogen of Scots pine (P. sylvestris) in pathogenicity tests (Lilja et ai, 1992). The aim of this work was to further characterize the uninucleate Rhizoctonia sp., which has been commonly isolated from roots of Finnish nursery grown conifer seedlings, together with the isolate from P. Abies in Norway (Venn et ai, 1986). MATERIALS AND METHODS Fungal isolates Information on the geographic origin of the isolates studied, host species and seedling type, year and source are given in Table 1. The Finnish isolates were originally isolated on distilled water agar (DWA) (1%) from surface sterilized root pieces as described by Lilja et ai. (1992). Anamorphic characteristics Hyphal characteristics (growth pattern, hyphal diameter, septal structure and the number of nuclei per cell) were examined after growth of the isolates on microscope slides coated with low strength (1/8) potato dextrose agar (PDA) (4-88 g PDA and 13'12 g agar (Difco laboratories, USA) I_l1 _1 H 2 O) that were maintained in a moist atmosphere for 48 hat 24 °C. All the following hyphal and basidial dimensions were measured using a light microscope equipped with a stage and eyepiece micrometer. The diameter of 15 subapical cells of main runner hyphae were randomly measured from four colonies of each isolate at 400 x magnification. The septal condition of hyphae was examined under phase contrast at 1000 x magnification. Nuclei were stained with HCI-Giemsa following the fixing and staining procedures described by Wilson (1992), and numbers in tip and subapical cell pairs (100 cells each) were counted at 400 x magnification. For characterization of general cultural features, petri-dishes containing PDA were centrally inoculated by transferring a 5x5 mm block from the margin of an actively growing colony and incubated in the dark at 24°. Colony colour and the distribution size, shape, colour and structure of sclerotia for each isolate were regularly examined over a period of 21 d. The colours were designated using the mycological colour chart of Rayner (1970). Growth rates Five replicate PDA plates, similarly inoculated (as above) with each isolate were incubated in the dark at 7, 14, 18, 21, 24, 28 and 31°. The colony diameter was measured at 24 h intervals along two right angled axes. Hyphal anastomosis Hyphal anastomosis was microscopically examined on the surface of distilled water agar (DWA) (15 g agar 1 _1 ) in petri dishes to determine anastomosis groupings (Parmeter, Sherwood & Piatt, 1969) and on microscope slides coated with 1/8 PDA for detailed identification of the type of hyphal fusion reaction (Matsumoto, Yamamoto & Hirane, 1932; Yokoyama, Ogoshi & Ui, 1983; Yokoyama & Ogoshi, 1986). Isolates were paired in all combinations at a distance of 2 cm by inoculating the agar surface using a modified Pasteur pipette (Korhonen & Hintikka, 1980) producing small cylindrical inoculum plugs (2 mm x 1 mm diam.). Pairings were incubated at 21° until the margins of opposing colonies began to overlap. Hyphal anastomosis on DWA was directly observed through the petri-dishes at 100 x and confirmed at 400 x magnification. The frequency of perfect and imperfect hyphal fusions on 1/8 PDA was recorded from a total of 75 contact points, where in each case two opposing hypha either fused, crossed or grew in a juxtapositioned manner (Sneh etai, 1991), using phase contrast at 400 x magnification. All pairing combinations were made on two separate occasions. Two isolates, 263 and 264, were also paired in all combinations on DWA with the binucleate Rhizoctonia (Ceratobasidium spp.) tester isolates (AG-A to AG-S, deposited in the ATCC) (Sneh et al., 1991), R. alpina (CBS 309.35) and R. quercus (CBS 313.35) to identify any anastomosis reactions. Teleomorphic characteristics A new method utilizing living Scots pine seedlings to induce the perfect state was developed. Sterile Scots pine seedlings Isolate number Location Coordinates Host Seedling type Year Source 250 Lapinlahti, Finland 63° 21' N, 27° 24' E Picea abies Containerized 1992 A. Lilja 256 Jomala, Finland 60° 09' N, 19° 57' E Larix sibirica Containerized 1991 A. Lilja 260 Lapinlahti, Finland 63° 21' N, 27° 24' E Pirtus sylvestris Bare-rooted 1989 A. Lilja 263 Lapinlahti, Finland 63° 21' N, 27° 24' E Pinus sylvtslris Bare-rooted 1987 A. Lilja 264 Lapinlahti, Finland 63° 21' N, 27° 24' E Pinus sylvestris Bare-rooted 1989 A. Lilja 83-111/1N* Namsos, Norway 64° 29' N. 11° 29' E Picea abies Containerized 1983 K. Venn" * Venn et al. (1986). '■ Norwegian Forest Research Institute. Characterization of a uninucleate Rhizortonia sp. 1046 were prepared by first imbibing seeds in distilled water at 5° for 36 h and then washing in a Tween-80 solution (3 drops per 100 ml distilled water) for 5 min. After three separate rinses in distilled water the seeds were treated with hydrogen peroxide (30%, v/v) for 15 min and then washed in three changes of sterile distilled water. The seeds were plated on 1*2% water agar, 15 to 20 seeds per petri-dish, and incubated inverted at 21° in dark for 14 d. Three sterile seedlings were then aseptically transferred to each petri-dish containing 20 ml sterile distilled water. A 5 x 5 mm block from the margin of a 3-d-old colony of an isolate grown on PDA at 21° was transferred to the petri-dish which was then incubated on a laboratory bench with natural indirect lighting. Between 3 and 5 replicate petri-dish culture systems per isolate were prepared in separate experiments over the whole year. Following the development of hymenia on the water and seedling surface, determined visually and at 40 x magnification, samples were transferred to microscope slides and squash preparates made for measurements of basidial and basidiospore dimensions. RESULTS Anatttorphic characteristics All cultures showed the general Rhizoctonia hyphal characteristics. Basally constricted branches arose at acute angles behind the apices of the advancing hyphae and at right angles in the older hyphal regions. A dolipore septum was always formed near the point of origin of the branch. The mean width of subapical cells of the main runner hyphae ranged from 6*3 to 7-2 um (Table 2). All the counted tip and subapical cells of isolates 250, 264 and 83-111/ IN were uninucleate (Fig. 1 A). The percentages of binucleate (tip: subapical) cells in 256, 263 and 260 were 4:4, 1:3 and 11:11%, respectively. In the latter isolate, hyphae containing many consecutive binucleate cells originating from uninucleate hyphae (Fig. 1 B) were mainly restricted to small areas in the central parts of the colony. The isolates showed almost uniform cultural morphology on PDA. Buff coloured, velvety-looking, young colonies grew in a radial manner and no zonation was observed. Hazel coloured spots first appeared on the central surface of the colony within 5 days and later appeared over the whole surface area to give a spotted appearance. Some of these very characteristic pigmented spots continued to spread covering an area of several mm 2 after an incubation period of 21 d. Within 14 d the first submerged, usually rounded sclerotia appeared. The individual submerged, but occasionally surface Table 2. Subapical cell widths of main runner hyphae* and dimensions of monilioid cells of sclerotia1* (|im) Fig. 1. Nuclei of isolate 260 stained with HCI-Giemsa. Hyphae with (A) only uninucleate cells and (B) a uninucleate hypha giving rise to a sidebranch with binucleate cells (arrowed). Bars = 10 pm. Isolate Hyphal width+ S.D. Monilioid cell Length Width 250 6-6 ±0-56 16-29 13-20 (5-4-7-7) 256 6-4 ±0-54 16-30 11-18 (5-4 ±7-8) 260 7-2 ±0-51 18-35 11-19 (5-8-8-4) 263 6-7 ±0-63 18-32 11-20 (5-2-7-8) 264 6-5 ±0-60 17-34 11-18 (4-8-7-7) 83-Ill/lN 6-3 ±0-57 14-32 10-17 (5-0-7-5) * 60 measurements (15 measurements from four separate colonies). b 60 measurements. A. M. Hietala, R. Sen and A. Lilja 1047 Fig. 2. The effect of temperature on the growth of different isolates on potato dextrose agar. ■ —■, 250; ￿—￿. 256; ©—©, 260; ￿—￿, 263; ￿—￿, 264; +—+ , 83-111/ IN. Fig. 3. Petri-dish/Scots pine fruiting system. Note white hymenial clusters (arrowed) of isolate 260 on the water surface after incubation of two weeks. located, sclerotia were fulvous to umber in colour with diameters up to 900 pirn. These submerged sclerotia tended to aggregate to form several mm large cauliflower-like structures after 21 d or longer incubation periods. The sclerotia were constructed of doliform to subglobose monilioid cells that were not organised into a rind and medulla. The dimensions of monilioid cells are presented in Table 2. Growth rates All isolates had very similar growth rates up to 24° on PDA, at 28° it was possible to separate some of the isolates (Fig. 2). Maximum growth rates of two isolates, 260 and 83-111/ IN, were at 21° and the others at 24° and all were unable to grow at 31°. Hyphal anastomosis The hyphae of all the uninucleate isolates anastomosed with each other both on DWA and thin films of 1/8 PDA. In self pairings on the latter agar, opposing hyphae often showed varying degrees of hyphal attraction which was followed by cell wall contact either between tips, a tip and a hyphal side wall or via bridging between adjacent side walls. Following contact a positive anastomosis reaction resulted in a perfect fusion which involved the loss of cell walls at the fusion junction and a maintenance to cytoplasmic continuity which occurred in 95-100% of the fused cells in all pairings. In non self pairings, similar hyphal pre-contact interactions were followed by imperfect fusions identified by a characteristic rapid sequence of events: cell wall dissolution, cytoplasmic granulation, a loss of cell turgor as detected by a narrowing of cell diameter and finally a complete vacuolation of between one and three cells on either side of the fusion junction, a phenomenon termed the killing reaction (Yokojama & Ogoshi, 1986). In most pairings, over 98% of the fusion cells were killed following anastomosis. The overall anastomosis fusion frequencies of self and non self pairings were in the range 49-57 and 45-71%, respectively. No hyphal fusions were recorded in pairings of 263 and 264 and either the binucleate Rhizoctonia isolates representing the known anastomosis groups of Ceratobasidium spp. (AG-A to AG-S) or R. alpina and R. quercus. Fig. 4. Typical basidia of the uninucleate Rhizoctonia sp. under phase contrast; isolates (A) 264 and (B) 83-111/ IN. Bars = 10 um. Characterization of a uninucleate Rhizoctonia sp. 1048 Table 3. Basidial" and basidispore1 ' dimensions (urn) of the uninucleate Rhizoctonia isolates Teleontorphic characteristics All the isolates, except 256, could be repeatedly fruited with the new method between April and August but not at other times of the year. The diurnal day and night room temperature fluctuations during the spring and summer varied between 21 and 30° but remained at a temperature of 20-22° during the autumn and winter. White coloured hymenium usually developed on the water surface near the margins of the petri dish but occasionally more centrally (Fig. 3) and also on the seedling surface. Basidia appeared within 8 to 15 d arising directly from basal hyphae or short side branches and the shape of metabasidia varied from obovate to subglobose (Fig. 4 A and B). The basidial and basidiospore dimensions are given in Table 3. The most frequent number of normally stout sterigmata was two and occasionally an adventitious septum was formed in the central or apical regions of the sterigmata. Branched sterigmata were also infrequently observed and in very rare cases unbranched sterigmata lacking basidiospores grew hundreds of urn long below the water surface, maintaining their characteristic width and forming regularly spaced adventitious septa. The basidiospores were ovoid to ellipsoid and germinated on the water surface by direct germ tube formation or in a minority of cases following repetition. DISCUSSION It is clear, from the anamorphic and teleomorphic data presented, that the isolates described represent a Rhizoctonia species. We have further examined the nuclear condition of over 30 isolates of similar cultural morphology, originating from conifer seedling roots taken from nurseries in Finland and Norway and they all contain predominantly uninucleate hyphae as described (unpublished data). It was not possible to compare our isolates with the uninucleate Rhizoctonia species described by Hall (1986) as the isolates were not deposited in a culture collection (G. Hall, pers. comm.). However, from his descriptions of the anamorph it is clear that these two species are not the same. No positive anastomosis reactions were detected in pairings of either 263 or 264 with the morphologically dissimilar isolates of R. alpina and R. quercus which had earlier been identified as being uninculeate by Burpee et al. (1980). In the data presented, only isolate 260 had binucleate cell numbers that were clearly higher than the expected frequencies explainable by random mitotic divisions (Tu, Kimbrough & Aldrich, 1977). The origin of occasional series of binucleate cells amongst the prevailing uninucleate cells in hyphae restricted to the central areas of the colony is not clear. Contamination of cultures can be ruled out as both nuclear types exist in the same hypha as shown in Fig. 1 8. The phenomenon does seem to be a general feature of our isolates as it has been observed to occur sporadically on various occasions under the same cultural conditions. Similar unpredictable changes may also explain the differences in nuclear condition reported for R. alpina and R. quercus (Burpee et al., 1980; Ogoshi et al.f 1983). We have also observed consecutive binucleate cells in hyphae of a colony grown out of a single uninucleate tip cell. Whether the two nuclei are identical, being brought together either by self anastomosis or as a result of loss of septal synchronization, or, more interestingly, are dissimilar possibly resulting from a reduction division requires further detailed genetic investigation. The very similar growth rates of all the isolates studied suggest that they are quite closely related, although at 28° it was possible to separate 260, 263 and 264 from the others. The uniformity of isolates was also further confirmed by the identification of a single anastomosis group. Within this group it is clear that the isolates represent different genotypes as non self anastomoses always resulted in the appearance of a killing reaction in neighbouring cells on either side of the point of hyphal fusion. Unsuccessful attempts to obtain the perfect state, made using the nutrient step-down (Adams & Butler, 1983 a, h) and antibiotic induction (Kangatharalingham & Carson, 1988) methods, prompted the development of the described method incorporating living seedlings of Scots pine. As no fruiting was observed on the water surface in the absence of seedlings, it is likely that the presence of plant material provides the fungus with a limited nutrient resource that may also actively induce fruiting through production of plant specific metabolites or breakdown products. During the autumn and winter months the centrally heated room temperatures remained very stable and thus the lack of a diurnal temperature gradient and inadequate natural lighting conditions may have contributed to the inhibition of fruiting. The method was reliable enough during spring and summer but the short window for fruiting is rather inconvenient and therefore requires further development. It was not possible to induce basidia of isolate 256, which had originally been isolated from larch ( Larix sibirica Ledeb.), in the petri-dish system containing P. sylvestris although 83-111/lN and 250 from P. Abies did fruit regularly. It may be the case that fruiting of 256 is host Metabasidium Sterigma Width Number Basidiospore Isolate Length Width Basal width Width/b.w. Length 1 2 3 4 Length Width 250 13-4 (10-7-14-9) 10-8 (9-3-12-1) 5 0 (3-4-6-2) 2-2 (1-6-31) 13-9 (10 0-25-8) 2-7 (1-8-3-8) 7 12 1 10-5 (96-13-4) 6-8 (5-3-7-6) 260 14 0 (12-0-16-4) 11 1 (100-12-3) 5 0 (3-4-6-7) 2-3 (1-8—3-1) 14-6 (9-4-44 0) 2-6 (2 0-3-6) 2 18 11-4 (96-13-4) 7-0 (5-7-8-6) 263 13-9 (12 0-16-3) 11-3 (10 0-13 0) 4-9 (3-9-7-7) 2-3 (1-7-2-9) 14-2 (7-0-20-3) 3 0 (1-9—4-8) 6 8 4 2 11-6 (96-14-3) 6-9 (5-3-8-4) 264 13-6 (11-1-17-0) 11 1 (8-7-13-4) 5 0 (3-1-6-8) 2-4 (1-6-3-6) 15-9 (8-7-27-2) 2-9 (1-8—4-4) 4 13 3 11-1 (8-6-14-8) 6-9 (5-1-8-6) 83-111/1N 15-7 (13-1-19-9) 12 0 (10-0-14-0) 5-2 (2-8-7-9) 2-5 (1-5-4-6) 16-8 (9-5-28-6) 3-2 (2-2-4-6) 1 14 5 11-6 (9-3-14-3) 6-8 (5-1-7-9) a Measurements from 20 basidia. '* Measurements from 40 basidiospores. A. M. Hietala, R. Sen and A. Lilja 1049 specific but further work including other larch isolates is needed to confirm this. On the basis of basidial characteristics, these isolates can be placed in Ceratobasidium (Talbot, 1965). The ratio between the widths of metabasidia and their supporting hyphae was generally over two, which is a major feature defining the genus. The development of basidia on basal hyphae or on short side branches and the repetitive germination of basidiospores are also typical of Ceratobasidium species. In contrast, there was a lack of affinity between two repre sentative uninucleate isolates (263 and 264) and tester isolates of known Ceratobasidium anastomosis groups AG-A to AG-S. The differing cultural morphology of these testers also indicates that they were not related to this uninucleate fungus. There is no description of similar anamorphs that could be related to these Nordic isolates in the anamorph keys of Ceratobasidium species summarized by Sneh et al. (1991). The teleomorph of our uninucleate isolates does seem to resemble that of C. bicorne J. Erikss. & Ryvarden, described from Danish field material, parasitic on the moss Polytrichum attenuatum (Brid.) {Polytrichastrum formosum (Hedw.) G. L. Sm.) (Eriksson & Ryvarden, 1973). The basidia of C. bicorne are obovate to subglobose (15-20 x B—lo nm) with two (in one case three) large and stout sterigmata (length, 12-18 pm; basal width, 3 pm). Basidiospores were narrowly ovoid to narrowly ellipsoid or subcylindrical (13-16 x 6-8 \xm). Since C. bicorne is known only from the type location, the basidial dimensions given above are not necessarily completely representative in terms of the intraspecific variation of this species, but allowing for this variation in the uninucleate isolates they could well be C. bicorne (L. Ryvarden, pers. comm.). For confirmation it would be necessary to isolate this fungus and obtain information on the nuclear condition, cultural characteristics and anastomosis reaction with the uninucleate isolates. The sexuality of the described uninucleate Rhizoctonia is at present being further investigated. The presence of predominantly uninucleate cells in all these, and many other isolates, would suggest that they are monokaryons of heterokaryotic bi- or multinucleate fungi. However, no morphological evidence of heterokaryon formation (e.g. aerial tufted mycelium) has yet been detected between paired isolates or within and between their single spore progeny although evidence of vegetative incompatibility, as indicated by strong demarcation zones, between paired progeny and their parents, is common (unpublished data). The fact that these isolates can be frequently isolated from conifer roots only as uninucleate hyphae does not support the monokaryon hypothesis either. Therefore it is possible that the killing reaction between uninucleate Rhizoctonia isolates is a sign of somatic incompatibility between the vegetative stage of this fungus, analogous to the one between R. solani field isolates (Ogoshi, 1987; Adams, 1988). It has also been possible to induce fruiting of single basidiospore progeny using the described system, as has been achieved in a Ceratobasidium sp. (Parmeter, Whitney & Piatt, 1967). This may suggest primary homothallism but further analysis of the genetic condition of single basidiospore derived cultures using isozyme or DNA/RFLP markers are needed (see Adams, 1988). These uninucleate Rhizoctonia appear to be important pathogens in the Nordic countries, causing root die-back in nursery seedlings of a range of conifer species, and thus information on the host range, mode of infection, population genetics and sexuality of these fungi are a major priority. In conclusion, this uninucleate fungus does fit into Ceratobasidium but because of the unusual nuclear condition of the field isolates we would prefer to describe it as a uninucleate Rhizodonia sp. having a Ceratobasidium fruiting stage. We would like to thank Professors A. Ogoshi and L. Burpee for providing us with Ceratobasidium tester isolates, Professor K. Venn and Dr D. Borja for their Norwegian Rhizoctonia sp. and together with Professor L. Ryvarden, Dr K. Korhonen and Dr G.Hall for helpful discussions. A.H. and R.S. also thank The Natural Resources Research Foundation of Finland (Suomen Luonnonvarain Tutkimussäätiö) for funding this work. REFERENCES Adams, G. C. (1988). Thanatephorus cucumeris (Rhizoctonia solani) a species complex of wide host range. In Advances in Plant Pathology (ed. D. S. Ingram & P. H. Williams), Vol. 6, Genetics of Plant Pathogenic Fungi (ed. G. S. Sidu), pp. 535-552. Academic Press: London, UK. Adams, G. C. Jr & Butler, E. E. (1983 a). Influence of nutrition on the formation of basidia and basidiospores in Thanatephorus cucumeris. Phytopathology 73, 147-151. Adams, G. C. Jr & Butler, E. E (1983 b). Environmental factors influencing the formation of basidia and basidiospores in Thanatephorus cucumeris. Phytopathology 73, 152-155. Burpee, L. L., Sanders, P. L., Cole, H. Jr & Sherwood, R. T. (1980). Anastomosis groups among isolates of Ceratobasidium cornigerutn and related fungi. Mycologia 72, 689-701. de Candolle, A. P. (1815). Memoire sur les rhizoctones, nouveau genre de champignons qui attaque les racines des plantes et en particular celle de la luzerne cultivee. Memoires du Museum National d'Histoire Naturelle 2, 209-216. English, T. R., Ploetz, R. C. & Barnard, B. L. (1986). Seedling blight of long leaf pine by a binucleate Rhizoctonia solani-\ike fungus. Plant Disease 70, 148-150. Eriksson, J. & Ryvarden, L. (1973). The Corticiaceae of North Europe, Vol. 2, Aleurodiscus—Confertobasidium. Fungiflora: Oslo, Norway. Hall, G. (1986). A species of Rhizoctonia with uninucleate hyphae isolated from roots of winter wheat. Transactions of the British Mycological Society 87, 466-471. Huang, J. W. & Kuhlman, E. G. (1990). Fungi associated with damping-off of slash pine seedlings in Georgia. Plant Disease 74, 27-30. Kangatharalingham, N. & Carson, M. L. (1988). Technique to induce sporulation in Thanatephorus cucumeris. Plant Disease 72, 146-148. Korhonen, K. & Hintikka, V. (1980). Simple isolation and inoculation methods for fungal cultures. Karstenia 20, 19—22. Lilja, A., Lilja, S., Poteri, M. & Ziren, L. (1992). Conifer seedling root fungi and root dieback in Finnish nurseries. Scandinavian Journal of Forest Research 7, 547-556. Matsumoto, T., Yamamoto, W. & Hirane, S. (1932). Physiology and parasitology of the fungi generally referred to as Hypochnus Sasakii Shirai. I. Differentiation of the strains by means of hyphal fusion and culture in differential media. Journal of the Society of Tropical Agriculture 4, 370-388. Ogoshi, A. (1975). Grouping of Rhizoctonia solani Kuhn and their perfect stages. Review of Plant Protection Research 8, 93-103. Ogoshi, A. (1987). Ecology and pathogenicity of anastomosis and intraspecific groups of Rhizoctonia solani Kiihn. Annual Review of Phytopathology 25, 125-143. Characterization of a uninucleate Rhizoctonia sp. 1050 Ogoshi, A., Oniki, M., Araki, T. & Ui, T. (1983). Studies on the anastomosis groups of binucleate Rhizoctonia and their perfect states. Journal of the Faculty of Agriculture, Hokkaido University 61, 244-260. Parmeter, J. R. Jr. Sherwood, R. T. & Piatt, W. D. (1969). Anastomosis grouping among isolates of Thanatephorus cucumeris. Phytopathology 59, 1270-1278. Parmeter, J. R. Jr & Whitney, H. S. (1970). Taxonomy and nomenclature of the imperfect state. In Rhizoctonia solani, Biology and Pathology (ed. J. R. Parmeter Jr), pp. 7-19. University of California Press: Berkeley, U.S.A. Parmeter, J. R. Jr, Whitney, H. S. & Piatt, W. D. (1967). Affinities of some Rhizoctonia species that resemble mycelium of Thanatephorus cucumeris. Phytopathology 57, 218-223. Rayner, R. W. (1970). A Mycological Colour Chart. Commonwealth My cological Institute: Kew, U.K. Roll-Hansen, F. & Roll-Hansen, H. (1968). A species of Rhizoctonia DC. ex Fr. damaging spruce plants in nurseries in Southern Norway. Meddelelser fra Det Norske Skogforsoksvesen 82, 421-440. Saksena, H. K. & Vaartaja, O. (1961). Taxonomy, morphology and pathogenicity of Rhizoctonia species from forest nurseries. Canadian Journal of Botany 39, 627-647. (Accepted 8 February 1994) Sneh, B.( Burpee, L. & Ogoshi, A. (1991). Identification of Rhizoctonia Species. The American Phytopathological Society: St Paul, U.S.A. Talbot, P. H. B. (1965). Studies of 'Pellicularia' and associated genera of Hymenomycetes. Persoonia 3, 371-406. Tu, C. C, Kimbrough, James W. & Aldrich, H. C. (1977). Cytology and ultrastructure of Thanatephoms cucumeris and related taxa of the Rhizoctonia complex. Canadian Journal of Botany 55, 2419-2436. Vaartaja, O. & Cram, W. H. (1956). Damping-off pathogens of conifers and of Caragana in Saskatchewan. Phytopathology 46, 391-397. Venn, K., Sandvik, M. & Langerud, B. R. (1986). Nursery routines, growth media and pathogens affect growth and root dieback in Norway spruce seedlings. Meddelelser fra Norsk Institutt for Skogforskning 39, 313-328. Wilson, A. D. (1992). A versatile giemsa protocol for permanent nuclear staining of fungi. Mycologia 84, 585-588. Yokoyama, K. & Ogoshi, A. (1986). Studies on hyphal anastomosis of Rhizoctonia solani. IV. Observation of imperfect fusion by light and electron microscopy. Transactions of the Mycological Society of Japan 27, 399-413. Yokoyama, K., Ogoshi, A. & Ui. T. (1983). Studies on hyphal anastomosis of Rhizoctonia solani. I. Observation of perfect fusion with light microscopy. Transactions of the Mycological Society of Japan 24, 329-340. II Eur. J. For. Path. 25 (1995) 136-144 © 1995 Blackwell Wissenschafts-Verlag, Berlin ISSN 0300-1237 Finnish Forest Research Institute, Vantaa, Finland Uni- and binucleate Rhizoctonia spp. Co-existing on the roots of Norway-spruce seedlings suffering from root dieback A. M. Hietala Summary Rhizoctonia fungi were isolated from the roots of 2-year-old nursery-grown Norway-spruce seedlings displaying root-dieback symptoms. The most frequently isolated species, a uninucleate Rhizoctonia sp., was found to co-exist with binucleate Rhizoctonia in the same root system of several seedlings. All the uninucleate isolates anastomosed with each other forming a single anastomosis group with common cultural characteristics. Binucleate Rhizoctonia isolates were divided into several, morphologically dissimilar anastomosis groups (AG-I, R. spp.). In a pathogenicity test under sterile conditions, isolates belonging to the uninucleate Rhizoctonia sp. infected all root regions, particularly the root tips, resulting in a stunted root-system morphology, as was also observed in the isolation material. Binucleate Rhizoctonia spp. colonized only basal root regions, occasionally infecting cortical cells with monilioid hyphae, ana had no effect on root growth. 1 Introduction Since the first description of a Rhizoctonia species, R. crocorum (Pers.) DC: Fr., nearly 100 further Rhizoctonia species have been described (PARMETER and WHITNEY 1970). The genus encompasses a heterogeneous group of fungi with diverse relationships. PARMETER and Whitney (1970), and later Ogoshi (1975), therefore established criteria which restrict the genus Rhizoctonia to imperfect states of basidiomycetes. Several basidiomycete genera, e.g. Thanatephorus and Ceratobasidium, possess the Rhi zoctonia vegetative state. Since induction of fruiting of Rhizoctonia species in culture is often difficult, the identification of Rhizoctonia isolates has traditionally been based on anamorphic characteristics. However, the vegetative characteristics of some Rhizoctonia species overlap considerably, which leads to difficulties in species identification. The dis covery of differences in the nuclear condition of field isolates of R. solani (multinucleate, genus Thanatephorus) and some Rhizoctonia anamorphs closely resembling them (binu cleate, genus Ceratobasidium) has reduced the confusion frequently associated with these fungi (PARMETER and WHITNEY 1970), and the introduction of anastomosis groupings, together with cytomorphology of hyphae and morphology of cultures, has placed the characterization of Rhizoctonia isolates on a more solid footing. Today, R. solani isolates are assigned to anastomosis (incompatibility) groups based on affinities for hyphal fusion with members of designated anastomosis groups (AG-1 AG-10 and AG-BI). In this way, Rhizoctonia species belonging to the genus Ceratobasidium have been grouped into 21 anastomosis groups (AG-A AG-S; Sneh et al. 1991). In Norway, root dieback of Norway spruce (Picea abies (L.) Karst.) seedlings has caused considerable losses in nursery production (Venn et al. 1986). The symptoms of the disease are needle discolouration, partial or total death of the root system, and stunted growth. A similar disease has occurred on Norway-spruce and Scots-pine {Pinus sylvestris L.) seedlings in Finnish forest nurseries (Lilja et al. 1992). A Rhizoctonia sp. has been found to be U. S. Copyright Clearance Center Code Statement: 0300—1237/95/2503—0136 $ll.OO/0 Rhizoctonia spp. on Norway-spruce roots 137 involved with the disease in Norway and Finland (VENN et al. 1986; Lilja et al. 1992; LILJA 1994). Anastomosis pairing has confirmed that these Norwegian and Finnish isolates causing root dieback represent the same uninucleate Rhizoctonia sp., which, on the basis of basidial morphology, belongs to genus Ceratobasidium (Hietala et al. 1994). Previous reports on the pathogenicity of Rhizoctonia spp. associated with root dieback of conifer seedlings (Venn et al. 1986; Lilja et al. 1992; LILJA 1994) have not included information on the vegetative characteristics, anastomosis groups, or mode of infection of the Rhizoctonia isolates studied. The aim of this study was to obtain information on the identity, patho genicity and mode of infection of Rhizoctonia spp. isolated from Norway-spruce seedlings displaying root-dieback symptoms. 2 Material and methods 2.1 Isolates Containerized 2-year-old Norway-spruce seedlings suffering from root dieback were col lected from a nursery in southern Finland (Lamppi: 61°39' N, 22°44' E) in autumn 1992. Fungal isolations were obtained from the roots of 47 diseased seedlings as follows: 3-mm long pieces were cut from different parts of the root system displaying root-rot symptoms, washed under tap water, surface sterilized for 1 min in 0.5% sodium hypochlorite, rinsed three times with sterile distilled water, and transferred onto potato-dextrose agar (PDA) or 1.2% water agar. Plates were incubated at room temperature and examined daily. Mycelia typical of the form-genus Rhizoctonia were transferred onto PDA and grown for 21 days at 21 °C in darkness. A total of 145 Rhizoctonia isolates were obtained from 40 seedlings. They were grouped according to their cultural morphology on PDA and by screening of anastomosis reactions. Representative isolates from each group were chosen for further characterization. Since some of the original cultures contained sectors from which two Rhizoctonia types could be isolated, the representative isolates were purified by isolating a tip from a primary hypha (KORHONEN and HINTIKKA 1980). Two strains, previously isolated from nursery-grown Norway-spruce seedlings, des ignated 249 (Kerimäki: 61 °s2' N, 29°03' E) and 250 (Lapinlahti: 63°21' N, 27°24' E), were also included in further studies. Isolate 250 was included in a paper describing a uninucleate Rhizoctonia sp. (Hietala et al. 1994). The isolates used in this study are listed in Table 1. 2.2 Vegetative characteristics For establishment of hyphal characteristics, isolates were grown on 1/8 PDA (4.88 g PDA and 13.12 g agar/1 H 2O) coated slides at 21 °C for 48 h (Hietala et al. 1994). The diameter of 15 subapical cells of the main runner hyphae were examined from four colonies at 1000 x magnification under a light microscope for each of the investigated isolates. The septal structure was examined under phase contrast. Nuclei were stained according to the pro cedures described by Wilson (1992). Colonial characteristics were determined from five replicate PDA plates of each isolate, which had been centrally inoculated and incubated at 21 °C in darkness for 21 days. The colours were designated according to RAYNER'S (1970) mycological colour chart. The colony diameter was measured every 24 h along two axes at right angles. 2.3 Hyphal anastomosis Hyphal anastomosis was microscopically examined on 2% water agar to determine anas tomosis groupings. Isolates were paired in all combinations at a distance of 1.5 cm by 138 A. M. Hietala Table 1. Isolate characteristics and the effects of inoculation with different isolates on the root growth of Norway-spruce seedlings. Uni- and binucleate isolate pairs 83-L/83-J, TB-A/TB-B and 811-K/811-L were originally isolated from the same seedlings 1 Morphological grouping is based on colonial characteristics on PDA after 21 days growth in darkness at 21 °C 2 Anastomosis group of a uninucleate Rbizoctonia sp.. Tester strain was isolate 250 (Hietala et al. 1994) 3 Isolates anastomose with Finnish strains belonging to the same morphological group, but not with the tester strains (AG-A-AG-S) of genus Ceratobasidium 4 Values are the means of 20 (control) or 10 (fungal inoculation) replicates. Means followed by the same lower-case letter in each column are not significantly (p = 0.05) different from each other using Tukey's HSD test 'Treatments are grouped on the basis of isolate nuclear condition. Means followed by the same upper-case letter in each column are not significantly (p = 0.01) different from each other using Tukey's HSD test Isolate characteristics Roo -growth indices after incubation Morphological' Nuclear Anastomosis Total root length Main-root length Lateral-root length Longest lateral root Strain no group condition group (cm) (cm) (cm) (cm) No. of root tips B3-L 1 uninucleate UAG 2 9.8"' 4 7.0 C 2.7 bc i.r d 9.9" T8-A 1 uninucleate UAG 2 9.1' 6.0 C 3.1 bc 0.4 d 11.6" Bll-K 1 uninucleate UAG 2 8.7 c 6.8° 1.8° 0.4 d 10.0" 249 1 uninucleate UAG 2 1 3.5 bc 7.0 c 6.5" bc 1.6 bcd 18.2' 250 1 uninucleate UAG 2 13.3 bc 7.9 C 5.4" be i.r d 15.9" mean 10.9 85 6.9® 3.9" 0.9" 13. 1 B T8-B 2 binucleacte AG-I 24.2" jl 9" b c 12.3' 4.3" b < 20.2' T5-Y 2 binucleate AG-I 27.3" 1 4.5 ab 12.9" 5.2" b 20.5' B3-J 3 binucleate 22.6" b 11.7' bc 10.9" b 3.8' bcd 20.5' B7-I 3 binucleate } 28.0" 16.8" ll.l" b 5.4" 15.8" Tl-C 4 binucleate 5 24.1" 14.1" b lO.O" 1 * 4.1" bc 18.7' Bll-L 5 binucleate > 22.2* b 1 0.5 bc 12.0" 20.2' mean 24. 7 A 13. 3 a 11. 5 a 4.5 a 19.3 A control 26.1" a 14.1 " bA 11.9" a 4 4^bA 20.3' A Rhizoctonia spp. on Norway-spruce roots 139 inoculating the agar using a modified Pasteur pipette (Korhonen and Hintikka 1980). Pairings were incubated at 21 °C until the margins of opposing colonies began to overlap. Hyphal anastomosis was scanned at 100 x and confirmed at 400 x under a light microscope. Binucleate isolates were also paired with binucleate Rhizoctonia ( Ceratobasidium spp.) tester isolates (AG-A to AG-S, deposited in the ATCC; Sneh et al. 1991). 2.4 Pathogenicity The pathogenicity of isolates was tested under sterile conditions. Norway-spruce seeds were surface sterilized with 30% H 2 O, as described by Hietala et al. (1994). Sterilized seeds were plated on 1.2% water agar and incubated at 21 °C in the dark until germination. Germinated seedlings were transferred to 55 ml test tubes containing 20 ml fertilized peat (ST4OO Finnpeat, Satoturve Oy, Finland) commonly used in forest nurseries. The peat had been sterilized by gamma irradiation (5 Mrad, 72 h) and moistened to a level of 40% (w/v). Tubes were scaled with sterile cotton plugs, weighed, and transferred to a green house, where the test was performed between June and August under natural lighting. During the test, the moisture of the peat was kept constant by weighing and watering each tube with sterile distilled water three times a week. The seedlings were inoculated at an age of 5 weeks with a 5 x 5 mm block taken from the margin of an actively growing PDA colony; 10 seedlings were inoculated with each isolate. As a control, 20 seedlings were inoculated with a sterile 5x5 mm PDA block. Seedlings were harvested at an age of 9 weeks, their roots were washed clean under tap water, and each root system was photocopied. The length of the roots was measured with a map measurer and the number of root tips was counted. Four root systems from each treatment were stained with trypan blue according to the procedures described by PHILLIPS and HAYMAN (1970). Stained root systems were stored in lactic acid at room temperature in darkness and examined for surface hyphae and infection with a light microscope at 40-400 x magnification. The analysis of variance and Tukey's HSD test were used for statistical analysis. 3 Results 3.1 Vegetative characteristics and hyphal anastomosis All examined isolates showed Rbizoctonia characteristics. Basally constricted hyphal bran ches arose near the distal septum of cells. A dolipore septum was formed near the point of origin of the branch. No rhizomorphs, clamp connections, or conidia, except for monilioid cells, were observed. The sclerotia were constructed of monilioid cells that were not organ ized into a rind and medulla. The isolates could be grouped into five groups on the basis of cultural morphology and anastomosis reactions (Table 1). Of the 145 isolates, 73 (obtained from 32 seedlings) had uninucleate cells and common cultural characteristics, and the remaining 72 isolates (obtained from 25 seedlings) were quite evenly distributed within four binucleate, mor phologically dissimilar groups. A total of 18 seedlings hosted both a uni- and a binucleate Rbizoctonia group; all the four binucleate groups were represented in these seedlings. In one seedling, isolates belonging to two binucleate groups could be found together with a uninucleate Rbizoctonia. When the seedling hosted more than one Rbizoctonia type, they were almost always isolated from different root segments. Isolates 83-L (code: seedling isolation), TB-A, 811-K, 249 and 250 were uninucleate, with mean hyphal diameters of 5.7-6.3 /fm (range of individual measurements: 5-8 /im) and a diametric growth rate of 13.5-15.0 mm/24 h. Culturally, the isolates were practically identical to the reference isolate 250: distinctive, small, hazel-coloured regions within the otherwise buff-coloured surface hyphae gave cultures a spotted appearance (HIETALA ct al. 140 A. M. Hietala 1994). Surface-located and submerged, and fulvous to umber-coloured sclerotia were readily formed. All isolates anastomosed with each other producing a killing reaction, as described by Yokoyama and OGOSHI (1986) for R. solani isolates. Isolates did not anastomose with the binucleate Finnish isolates tested. Isolates T5-Y and TB-B were binucleate, with a hyphal diameter of 5.8-6.3 /(m (range 5- 7 /im) and a growth rate of 13.1-15.4 mm/24 h. Isolates had white-to-cream-coloured surface hyphae with strong zonation. Isolate TB-B also had aerial tufts of monilioid hyphae. These two isolates anastomosed with each other, and with the culturally similar AG-I (tester isolate ATCC 76143), producing a killing reaction. Isolates 83-J and 87-I were binucleate, with a hyphal diameter of 5.2-5.4 fim (range 4-6 /im) and a growth rate of 11.8-13.2 mm/24 h. Surface and aerial mycelium were buff coloured and both isolates formed a few embedded, isabelline-coloured sclerotia. Isolate 87-I also readily formed buff-to-isabelline-coloured surface sclerotia. Isolates anastomosed with each other producing a killing reaction. Isolates did not anastomose with any of the tester strains of the genus Ceratobasidmm. Isolate Tl-C was binucleate, with a hyphal diameter of 5.1 /im (range 4-6 /im) and a growth rate of 12.6 mm/24 h. Culturally, the isolate was quite different from other examined groups; the buff-to-rosy-buff-coloured surface mycelium was strong and continuous in the centre of the colony. Surface sclerotia were primrose; embedded sclerotia almost orange. Tl-C did not anastomose with any of the Finnish isolates belonging to another mor phological group, nor with any of the tester isolates. Isolate 811-L was binucleate, with a hyphal diameter of 5.6 fim (range 5-7 fim) and a growth rate of 14.2 mm/24 h. The velvet-like, white-to-buff-coloured surface mycelium was centrally zonated. No sclerotia or aerial tufts of monilioid cells were formed. 811-L did not anastomose with any of the Finnish isolates belonging to another morphological group, nor with any of the tester isolates. 3.2 Pathogenicity test Staining of sampled root systems showed the presence of Rhizoctonia hyphae on all inocu lated roots. However, the seedlings survived in all treatments during the experiment. Roots inoculated with uninucleate isolates were strongly pigmented and clearly stunted. The effect of inoculations on root growth is shown in Table 1. The roots inoculated with binucleate isolates were not statistically different from the control in any root terms. Seedlings inoculated with a uninucleate isolate clearly showed poorer root growth than the control seedlings and those inoculated with binucleate strains, and they differed statistically from the control seedlings in terms of total root length and length of the main root. When comparing the effect of uni- and binucleate strains isolated from the same seedling, the seedlings inoculated with uninucleate strains showed generally poorer root growth than the seedlings inoculated with the binucleate strains. This was most clearly shown in total root length; all the treatments inoculated with a uninucleate isolate differed statistically from the treatment inoculated with the corresponding binucleate isolate. When seedlings were grouped on the basis of the nuclear condition of the inoculated fungus, the uninucleate group was significantly different from the control and binucleate group in all root indices. The uninucleate isolates colonized the root surface along the whole root system and practically all root tips were infected by continuous mass of aggregations of short, branched hyphae and appressoria-like structures, often several hundred /im long (Fig. la, b). Similar aggregates, although considerably smaller, were also formed abundantly on other root areas infecting the cortex (Fig. lc). Focussing through the tissues to the centre of the root suggested that, in the root tips, uninucleate isolates grow in the vascular cylinder (Fig. id). In seedlings inoculated with binucleate isolates, roots were commonly free of hyphae up to several mm behind the root tip. Compared to treatments inoculated with uninucleate Rhizoctonia spp. on Norway-spruce roots 141 Fig. 1. Root infection by isolates TB-A (uninucleate Rhizoctonia sp., a-d) and TB-B (AG-I, e, f) that were originally isolated from the same root system; a. A lateral root tip typically covered with hyphae (bar 50 /im); b. Hyphai aggregates on the surface of a lateral root tip (bar 20 /5 mm No. of 2nd- No. of proliferating Host Treatment* T cm g g cm cm f in length order laterals* 2nd-order laterals* P. ab ie s Control 0 4.3a 0.015a 0.05a 11.1a 2.5a 7.9a 1.9a Control 1 9.0 b 0.2496 1.576 41.7c 18.7c 23.7c 28.26 12.9a 248 1 7 Jab 0.209 ab 0.70a 24.36 8.36 17.66c 19.86 5.1a 255 1 1.3ab 0.150a6 0.43a 19.1a6 5.4a6 14.5a6 20.66 5.9a 256 1 8 .6b 0.23 6b 0.67a 17.3a6 5.8a6 15.96 20.96 6.1a P. sylvestris Control 0 3.9 a 0.015a 0.06a 14.9a 3.3a 8.1a 6.0a Control 1 10.3c 0.718c 2.91c 100.5c 25.1c 44.1c 25.3c 14.66 248 1 6.1b 0.5206 1.416 51.46 12.36 24.76 14.96 6.2 a 255 1 6.9b 0.311b 0.946 43.6a6 8.6a6 20.5a6 16.46 9.1a6 256 1 6.5 b 0.494 b 1.526 64.06 12.26 28.96 20.76c 9.5a6 L. sibirica Control 0 4.3 a 0.013a 0.09a 17.1a 3.9a 8.9a 4.7a Control 1 20.1 b 0.5166 1.986 54.96 12.1c 18.46 14.46 7.6a 248 1 19.5 b 0.4176 1.656 33.0a6 9.46c 16.16 13.16 8.0a 255 1 18 .6b 0.4506 1.696 39.1a6 7.86 17.16 17.66 7.4a 256 1 20.1b 0.5256 1.846 26.4a 8.56c 17.46 14.46 7.6a Note: Seedlings were inoculated at the age of 7 weeks ( T = 0) and harvested after 5 months incubation (7 = p = 0.01). •The strains shown in bold were originally isolated from the respective host. t Values represent the average length of the five longest first-order long laterals. ￿Parameters were recorded at a standard region (i.e., first 6 cm at the base of the long lateral). 1). Values (n = 15) followed by the same letter do not differ from each other (Tukey's HSD test, 474 Can. J. For. Res. Vol. 27, 1997 The primary root of all larch, pine, and spruce seedlings was actively growing at the time of inoculation, possessing a pointed tip with a well-developed root cap and showing no signs of a metacutization layer (see Wilcox 1954). The first protoxylem elements were observed 1500-3500 Jim behind the apical initials. Primary roots possessed the largest distance between the apical initials and the first xylcm elements within the root system of all seedlings. In six Norway spruce seedlings, 10-50% of the long laterals (>5 mm in length) had a rounded root apex with a structure identified as a metacutization layer, indicating ceased growth and dormancy. Similarly, 10-90% of long laterals of six Sibe rian larch seedlings had a rounded root apex and a metacuti zation layer (Fig. 1). In the remaining spruce and larch seedlings and in all 15 pine seedlings, all the long laterals had a pointed apex with a well-developed root cap and showed no signs of a metacutization layer. These were regarded as ac tively growing roots (Fig. 2). In the dormant long laterals of spruce and larch, the protoxylem elements differentiated usu ally 150-500 behind the apical initials, whereas in the growing long laterals of spruce, larch, and pine the protoxylem elements differentiated 700-2500 behind the apical initials. Similarly, the short laterals (<5 mm) of all host species could be divided into growing and dormant roots. In short laterals with a metacutization layer, the protoxylem commonly differentiated 100-200 behind the apical meristem. In the growing short laterals, protoxylem differentiation was ob served 200-500 behind the meristem. In pine and spruce, some of the growing short laterals, unlike the growing long laterals, had a rounded apex with a poorly developed root cap. These roots were regarded as short roots, whereas all the growing short laterals of larch had a pointed apex with a well-developed root cap. At this time, no mycorrhizal roots were observed on any tree species. Root system morphology and infection Rhizoctonia could be isolated from all inoculated seedlings, but not from the control seedlings. The Rhizoctonia isolates obtained from the inoculated seedlings showed somatic com patibility against the original, cold-room-stored culture. A so matic incompatibility reaction, formation of a demarcation line between the paired colonies, was produced when an obtained isolate was paired against the other two strains of Rhizoctonia used in this study. Visually, the root growth of spruce and pine seedlings was notably affected by the inoculation. The primary roots and many of the first-order long laterals were commonly broken with the tip missing, the remaining root tips were heavily pig mented, and the root system seemed considerably stunted. In inoculated larch seedlings, root tips of the primary roots and first-order long laterals, particularly at the basal part of the root system, were occasionally missing. In contrast with pine and spruce, no visually striking differences were observed in pig mentation or in the root system size compared with the control seedlings. Needle discolouration symptoms were not observed in any host species. Significant decreases (Tukey's HSD, p - 0.01) were observed among the root parameters of all inoculated trees (Table 1). No major differences were detected between seedlings inoculated with different isolates. The only significant decreases in the shoot length and dry weight were found for the inoculated seedlings of pine. In pine, the inoculated treatments were sig nificantly different from the control at 7* = 1 in all measured root parameters, excluding the two related to second-order lat erals. In spruce, the inoculated seedlings were different from the control at T = 1 in terms of root fresh weight, length of the primary root, length of the five longest first-order laterals, and with one exception, also in the number of first-order laterals exceeding 5 mm in length. In pine and spruce, there was a clear, although in most cases not significant, decrease in the root parameters relating to second-order laterals. In larch, a decrease in the root growth was evident in length of the pri mary root and in length of the five longest first-order laterals, but the difference to the control at T = 1 was not statistically significant in most cases. All the stained root systems of inoculated pine, spruce, and larch seedlings had relatively wide hyphae (4-8 }im) (see Hie tala et ai. 1994). These could be easily identified as Rhizoc tonia on the basis of the growth characteristics (branching near the distal septum of cells, formation of a septum in the branch near the point of origin, constriction of the branches, and ab sence of clamp connections, see Ogoshi 1975). Rhizoctonia like hyphae were not detected on the roots of uninoculated control seedlings; the observed fungi had narrow hyphae (1-3 nm) with different growth characteristics and often clamp connections. At the time of final harvesting, many pine and spruce seed lings in all treatments had some mycorrhizal root tips. In gen eral though, including the control seedlings, the level of mycorrhizal infection was low and mycorrhiza were localized. The associated mycorrhizal fungi were characterized by nar row (2-3 clamped hyphae. No mycorrhizal roots were observed in larch seedlings. Several collars indicating previous metacutization layers were commonly observed on the non mycorrhizal short roots of pine and spruce (Fig. 3). No differences were observed in the external growth of Rhizoctonia hyphae nor in the locality of infection areas on the roots of different hosts. On the root surface, excluding the tip region where protoxylem had not yet differentiated, the growth of Rhizoctonia hyphae was commonly sparse. Cortical cells were frequently infected by a short hyphal branch either di rectly or via an appressorium-like swelling. Occasionally, the fungus filled cortical cells with monilioid hyphae (Fig. 4), but the frequency of this type of infection was low and the phe nomenon was not observed in every stained root system. No Rhizoctonia hyphae were observed at the root collar region. In the tips of growing roots, aggregations of short-celled hyphae were commonly observed on the root surface (Fig. 5) and within the intercellular spaces of the outer cortex in the region where protoxylem elements had not yet differentiated. Further spread to the vascular cylinder by penetration hyphae, initiated underneath these hyphal aggregations, was both inter and intra-cellular (Figs. 6-7). Penetration hyphae were most frequently formed at the apical initials and 100-300 be hind them (Fig. 8). After this region, the frequency of penetra tion hyphae decreased gradually, and penetration hyphae were seldom observed after the protoxylem differentiation. At the apex of the root, penetration hyphae were commonly orien tated towards the apical meristem (Fig. 9). After reaching the vascular cylinder, the Rhizoctonia hyphae turned to grow to wards the base of the root (Fig. 10). © 1997 NRC Canada Hietala 475 Figs. 1-3. Unsectioned roots of uninoculated control seedlings cleared with KOH, bleached with alkaline H 20 2, and stained with trypan blue according to Koske and Gemma (1989). Scale bar = 40 Jim. Fig. I. A dark mctacutization layer at the apex of a dormant long lateral of Siberian larch. Note the differentiation of protoxylcm elements (arrow) close to the apical mcristcm. Fig. 2. The apex of an actively growing long lateral of Siberian larch. Fig. 3. A dormant non-mycorrhizal short root of Norway spruce with several basal collars (arrow) indicating previous mctacutization layers. In all stained root systems of pine and spruce, the majority of the first-order long laterals, including the primary root, were infected. In roots where the root tip was missing, protoxylcm elements with intracellular Rhizoctonia hyphae were fre quently protruding at the breakage point (Fig. 11). Young long laterals, probably adventitious roots, were commonly observed immediately behind the breakage point in all host species and in most cases, had been infected. In the most stunted root systems, several long laterals, ex cluding first-order roots, were free of Rhizoctonia hyphae and infection in the root tip region. The majority of the uninfected long laterals of all seedlings were dormant, whereas none of the infected long laterals had formed a metacutization layer. Occasionally, there was a small amount of surface hyphae at the apex of a dormant long lateral, but the hyphal growth was relatively sparse and no penetration hyphae were formed. No surface hyphae were observed on the root tips of the unin fected, growing long laterals. Compared with primary roots and long laterals, short roots were rarely infected by Rhizoc tonia even in those root systems of pine and spruce with no mycorrhizal root tips. The vast majority of short roots were free of Rhizoctonia hyphae and a mctacutization layer had been formed, and as in control seedlings, several collars indicating previous metacutization layers were commonly observed. Besides short roots, long laterals of pine and spruce were also occasionally mycorrhizal. In inoculated pine and spruce seedlings, dual infection of a common long lateral root tip by Rhizoctonia and a mycorrhizal fungus was occasionally ob served. The area with Rhizoctonia penetration hyphae was fol lowed by a Hartig net region. Compared with the stained root systems of pine and spruce, the infection level in inoculated larch seedlings was variable. In three of the six stained root systems, several laterals, par ticularly first-order roots, had been infected. In the other three seedlings, regardless of the origin of the inoculated isolate, the infection of root tips was very localized, restricted to a few © 1997 NRC Canada 476 Can. J. For. Res. Vol. 27, 1997 Figs. 4-14. Infection of roots by uninucleate Rhizoctonia sp. Unless otherwise stated, the infection characteristics are demonstrated on Norway spruce; no major differences were observed in the mode of infection on different hosts. Figures 4-5 and 8-11 represent observations on unsectioned roots treated according to the protocol of Koske and Gemma (1989). The rest of the figures (6-7 and 12-14) represent paraffin sections. The scale bar = 10 Fig. 4. Monilioid hyphae within cortical cells at the base of a long lateral. Fig. 5. Hyphal proliferation on the root surface at the apex of a long lateral. Note the bidirectional growth pattern (arrows), probably a result of hyphal anastomosis. Fig. 6. A hyphal aggregation (ha) under the epidermis at the apex of a long lateral and intracellular penetration towards the vascular cylinder ( vc ) filled with Rhizoctonia hyphae. Fig. 7. Intercellular hyphal aggregations (ha) within the cortex close to the apex of a long lateral. Note intercellular © 1997 NRC Canada Hietala 477 penetration under the hyphal aggregations (arrows). Fig. 8. Multiple penetration hyphac (examples shown with arrows) at the apex of a long lateral. Fig. 9. A hyphal aggregation (ha) on the root surface and penetration towards the apical meristcm (am). Fig. 10. A penetration hypha and basipctal growth within the vascular cylinder. Note the differentiation of the protoxylem (arrow). Fig. 11. Intracellular Rhizoctonia hyphae within protoxylem sticking out at a breakage point of a long lateral (Siberian larch). Fig. 12. A typically macerated vascular cylinder of an infected long lateral (Scots pine) filled with narrow hyphae of secondary fungi (sf). Rhizoctonia hyphae arc indicated with arrows. Fig. 13. Rhizoctonia hyphac (arrows) within the mctaxylem of a first-order long lateral. Fig. 14. Spread of Rhizoctonia hyphae (arrows) within the vascular cylinder of a young long lateral to the mother root. © 1997 NRC Canada 478 Can. J. For. Res. Vol. 27, 1997 Figs. 15-16. Hyphal growth of the uninucleate Rhizoctonia sp. under the membrane-isolated roots of Norway spruce and fruiting of the fungus in the presence of the host seedlings. Fig. 15. Proliferation of hyphae under the membrane-isolated root in a region where root growth has occurred (bar = 100 urn). Fig. 16. Hymenial clusters (arrows) on the water surface in the presence of Norway spruce (top), Siberian larch (right), and Scots pine (left) seedlings (bar = 10 mm). neighbouring laterals in a common mother root. In general, very few surface hyphae of Rhizoctonia were observed at the basal root regions in these root systems. No differences were observed in the hyphal growth within the vascular cylinder of different seedlings. The apical meristem and the neighbouring vascular cylinder were usually heavily macerated and were often colonized by secondary fungi with very thin hyphae (1-2 }im) (Fig. 12). Rhizoctonia hyphae spread within the vascular cylinder, particularly in the xylem elements and associated vascular parenchyma (Figs. 11-13). Hyphal growth was usually restricted close to the original infection site. At the breakage point of a first-order long lateral, where a relatively short (<1 cm) long lateral had been infected, Rhizoctonia hyphae occasionally spread within the vascular cylinder to the parent root (Fig. 14). Hyphal growth under the membrane-isolated root J 1'"'" fa'»"»' "' V »■«..«. M..V Hyphal growth was greatly stimulated under the growing root tips of all host species. First initials of hyphal proliferation were observed 48 h after the root was introduced to the system. After 7 days incubation, abundant aggregations had formed under the root in the region between the original and immedi ate position of the grown root apex (Fig. 15). Increased hyphal proliferation was also observed near the root collar. The hy phae were not able to penetrate through the membrane during the 10-day incubation period. Fruiting In the fruiting experiment, all the seedlings died within a few days of inoculation. All isolates produced the basidial stage in the presence of each host, but not in control Petri dishes with out seedlings. Fruiting occurred between 7 and 14 days after inoculation. The most abundant hymenial production was ob served, regardless of the inoculated strain, in the presence of Scots pine (Fig. 16). On pine the fruiting frequencies of the isolates 248, 255, and 256 were four, two, and one, respec tively. On spruce, these frequencies were five, two, two and on larch, three, three, and five, respectively. Discussion Somatic compatibility tests confirmed the presence of the in oculated strains on the roots at the time of harvesting. No Rhi zoctonia like hyphae were observed on the roots of stained control seedlings at the end of the experiment. This is not surprising, since Rhizoctonia spp. have not commonly been isolated from peat and other soil-free growth media. The re sults of Venn et al. (1986) implicated the supporting sand beds as an inoculum source for Rhizoctonia related to root dieback in container production. No differences were observed in the root system charac teristics of seedlings inoculated with different isolates, and there were no differences in the mode of infection on different seedlings. The apical region of primary roots and long laterals was the primary penetration route into the vascular cylinder, similar to that found by Farquhar and Peterson (1989) in Fusarium oxysporum f.sp. pini on growing primary roots of young red pine (Pinus resinosa Ait.) seedlings. In growing conifer roots, the endodermis close to the apical initials is still at the primary, nonsuberized stage (e.g., Wilcox 1954; Leshem 1974; Johnson-Flanagan and Owens 1985; Warmbrodt and Eschrich 1985; Kottke and Oberwinkler 1990). Cessation of root elongation results in the formation of a so-called metacu tization layer, where lignified and suberized cells connect the subsurface cells of the root apex to the suberized endodermis © 1997 NRC Canada Hietala 479 (e.g., Wilcox 1954). The isolated root cap and cortical cells die, which causes rounding of the root tip (Johnson-Flanagan and Owens 1985). At the time of inoculation, both larch and spruce seedlings had long laterals that, based on the presence of a mctacutization layer, were in dormancy. At the time of final harvesting, the majority of the uninfected long laterals of all host species were in dormancy. No infection structures had been formed when there were surface hyphae of Rhizoctonia at the apex of a dormant root. In contrast, no signs of a mctacu tization layer were observed in the infected root tips. These observations suggest that only actively growing roots are in fected by this pathogen. The prepenetration stage of the uninucleate Rhizoctonia sp. was characterized by formation of hyphal aggregates giv ing rise to penetration hyphae. Rhizoctonia solani has been shown to form similar hyphal aggregations, termed infection cushions, on several plants (e.g., Dodman and Flentje 1970). In addition, hyphal growth was notably stimulated under the membrane-isolated root tips. It is, therefore, hypothesized, that exudates from the growing root tips stimulate hyphal growth of this Rhizoctonia species and provide energy for the luxuri ous growth preceding the formation of penetration hyphae. Components of the root exudates of some conifer seedlings have been shown to stimulate hyphal growth of Rhizoctonia (Agnihotri and Vaartaja 1969) and sporangium germination of Pythium (Agnihotri and Vaartaja 1967). Moreover, there is considerable evidence from other hosts that plant exudates are essential for the formation of infection cushions of R. solani (e.g., Dodman and Flentje 1970; Armentrout et al. 1987). It should be noted that compared with primary roots and long laterals, short roots were seldom infected by the pathogen. In primary roots and long laterals, the likelihood of being in fected should be multiple, since they will encounter pathogen propagules and hyphae within large medium volumes. Short roots, on the other hand, are restricted in growth and thus con fined to a particular space. Spruce and pine seedlings showed considerably stunted root systems, while larch seedlings seemed less affected. However, the first-order long laterals of uninoculatcd larch seedlings showed considerably poorer growth capacity than those of spruce and pine, which undoub tly partially contributes to the relatively small difference in growth between the inoculated and control seedlings of larch. Unlike pine and spruce, considerable differences were ob served in the level of infection between individual larch seed lings. Wilcox (1954) found that individual long laterals of noble fir (Abies procera Rehd.) had several growth periods during the growing season, a single growth period of individ ual roots ranging from 2 to 6 weeks. Similar periodicity has been shown for roots of Picea glauca (Moench) Voss (Johnson-Flanagan and Owens 1985) and Pinus resinosa (Wil cox 1968). At the time of inoculation, some of the larch seed lings had a high percentage of dormant first-order long laterals. Unfortunately, the root growth patterns of different hosts and individual seedlings were not followed by sequential harvest ings after the inoculation. If only actively growing roots stimu late hyphal growth, the presence, frequency, and longevity of root dormancy at the time of inoculation could have a consid erable effect on the infection potential and the multiplying rate of the inoculum present in the system. At the basal parts of the roots, hyphal growth of the uninu cleate Rhizoctonia sp. was sparse and cortical cells were occa sionally colonized by monilioid hyphae. Saksena and Vaartaja (1961) showed that several binuclcatc Rliizoctonio species (sec Sneh ct al. 1991) associated with conifer seedlings were able to cause stunting of root systems. The infection was charac terized by massive production of monilioid hyphae within cor tical cells, but the authors do not mention whether the root tips or the vascular cylinder were infected by these fungi. Hietala (1995) examined the mode of infection of several binucleatc Rhizoctonia (AG-I, R spp.) coexisting with the present uninu cleate Rhizoctonia sp. in the roots of Norway spruce seedlings suffering from root dicback. The binuclcatc Rhizoctonia spp. in fected cortical cells with monilioid hyphae, at basal root regions only and had no effect on the root growth. Ferris et al. (1984) suggested that monilioid hyphae within host cells probably act as sclerotium-likc dispersal and survival units for Rhizoctonia. Over time, cortical cells will be sloughed off, the intracellular monilioid hyphae will be liberated into the growth medium, and if promoted by a nutritional stimulus (e.g., root exudates), they will germinate. In a previous study where the present uninucleate Rhizoc tonia species was initially characterized (Hietala et al. 1994), isolate 256 did not fruit on the used test plant, Scots pine. The success in fruiting this isolate on all hosts in the present study indicates that the process may be very sensitive. When single basidiospore isolates of a strain arc paired against each other, a somatic incompatibility reaction is commonly produced. Sin gle basidiospore isolates of this species can also be fruited, without mating, and the somatic incompatibility reaction is common among the second-generation offspring (A.M. Hie tala, K. Korhonen and R. Sen, unpublished). This indicates that this species is homothallic. Several genotypes, based on the somatic incompatibility reaction, have been found in a single forest nursery (A.M. Hietala, unpublished), which could imply that the pathogen fruits here. Genetically, this uninucleate Rhi zoctonia sp. is very homogeneous (Lilja et al. 1996), which would be a natural result of inbreeding. The possible interaction between Pythium and Rhizoctonia in root dieback disease has not been investigated. Excluding studies related to seedlings at the damping-off stage (e.g., Borja et al. 1995), the mode of infection of Pythium spp. associated to root dieback disease of conifer seedlings has not been studied, but as observed for this Rhizoctonia species, growing root tips are also regarded as the main target of Pythium spp. (Hendrix and Campbell 1973; Endo and Colt 1974). During the present study, the moisture of the growth medium was artificially kept at a constant level, but it was obvious that inoculated seedlings of pine and spruce needed gradually less watering than the control seedlings. Under nurs ery conditions, such a drastic reduction in the root biomass would result in moisture stress promoting fungal spread with zoospores. Further studies on the coexistence of these fungi can be justified. Acknowledgments The author is grateful to The Natural Resources Research Foundation of Finland (Suomen Luonnonvarain Tut kimussäätiö) for funding this work. Dr. Veikko Hintikka, Dr. Kari Korhonen, and Dr. Robin Sen arc thanked for their helpful criticism and Dr. Karen Sims, for revision of the Eng lish in the manuscript. © 1997 NRC Canada 480 Can. J. For. Res. Vol. 27, 1997 References Agnihotri, V.P., and Vaartaja, O. 1967. Root exudates from red pine seedlings and their effect on Pythium ultimum. Can. J. Bot. 45: 1031-1040. Agnihotri, V.P., and Vaartaja, O. 1969. Stimulation of Waitea circi nata by root exudates of Pinus cembroides. Can. J. Microbiol. 15: 1319-1323. Armentrout, V.N., Downer, A.J., Grasmick, D.L., and Wein hold, A.R. 1987. Factors affecting infection cushion development by Rhizoctonia solani on cotton. Phytopathology, 77: 623-630. Borja, 1., Sharma, P., Krekling, T., and Lönneborg, A. 1995. Cy topathological response in roots of Picea abies seedlings infected with Pythium dimorphum. Phytopathology, 85: 495-501. Dodman, R.L., and Flentje, N.T. 1970. The mechanism and physiol ogy of plant penetration by Rhizoctonia solani. In Rhizoctonia solani, biology and pathology. Edited by J.R. Parmeter, Jr. Uni versity of California Press, Berkeley, pp. 149-160. Endo, R.M., and Colt, W.M. 1974. Anatomy, cytology and physiol ogy of infection by Pythium. Proc. Am. Phytopathol. Soc. 1:215-223. Eriksson, J., and Ryvarden, L. 1973. The Corticiaceae of North Europe. Vol. 2. Aleurodiscus-Confertobasidium. Fungiflora, Oslo, Norway. Farquhar, M.L., and Peterson, R.L. 1989. Pathogenesis in Fusarium root rot of primary roots of Pinus resinosa grown in test tubes. Can. J. Plant Pathol. 11: 221-228. Ferris, R.S., McGraw, A.C., and Hendrix, J.W. 1984. Production of monilioid cells by binucleate Rhizoctonia isolates. Phytopathol ogy, 74: 867. Hall, G. 1986. A species of Rhizoctonia with uninucleate hyphae isolated from the roots of winter wheat. Trans. Br. Mycol. Soc. 87:466-471. Hendrix, F.F., and Campbell, W.A. 1973. Pythiums as plant patho gens. Annu. Rev. Phytopathol. 11: 77-98. Hietala, A.M. 1995. Uni- and binucleate Rhizoctonia spp. Co-existing on the roots of Norway spruce seedlings suffering from root die back. Eur. J. For. Pathol. 25: 136-144. Hietala, A.M., and Sen, R. 1996. Rhizoctonia associated with forest trees. In Rhizoctonia species: taxonomy, molecular biology, ecol ogy, pathology and disease control. Edited by B. Sneh, S. Jabaji- Hare, S. Neate, and G. Dijst. Kluwer Academic Publishers, Dordrecht, Netherlands, pp. 351-358. Hietala, A.M., Sen, R., and Lilja, A. 1994. Anamorphic and teleomor phic characteristics of a uninucleate Rhizoctonia sp. isolated from the roots of nursery grown conifer seedlings. Mycol. Res. 98: 1044-1050. Johnson-Flanagan, A.M., and Owens, J.N. 1985. Development of white spruce (Picea glauca) seedling roots. Can. J. Bot. 63: 456-462. Koske, R.E., and Gemma, J.N. 1989. A modified procedure for stain ing roots to detect VA mycorrhizas. Mycol. Res. 92: 486-488. Kottke, 1., and Oberwinkler, F. 1990. Comparative investigations on the differentiation of the endodermis and the development of the Hartig net in the mycorrhizae of Picea abies and Larix decidua. Trees, 4: 41-48. Leshem, B. 1974. The relation of the collapse of the primary cortex to the suberization of the endodermis in roots of Pinus halepensis Mill. Bot. Gaz. 135: 58-60. Lilja, A. 1994. The occurrence and pathogenicity of uni- and binu cleate Rhizoctonia and Pythiaceae fungi among conifer seedlings in Finnish forest nurseries. Eur. J. For. Pathol. 24: 181-192. Lilja, A., Lilja, S., Poteri, M., and Ziren, L. 1992. Conifer seedling root fungi and root dieback in Finnish nurseries. Scand. J. For. Res. 7: 547-556. Lilja, A, Hietala, A.M., and Karjalainen, R. 1996. Identification of a uninucleate Rhizoctonia sp. by pathogenicity, hyphal anastomosis and RAPD analysis. Plant Pathol. 45: 997-1006. Ogoshi, A. 1975. Grouping of Rhizoctonia solani KUhn and their perfect stages. Rev. Plant Prot. Res. 8: 93-103. Ogoshi, A., Oniki, M., Araki, T., and Ui, T. 1983. Studies on the anastomosis groups of binucleate Rhizoctonia and their perfect states. J. Fac. Agric. Hokkaido Univ. 61: 244-260. Saksena, H.K., and Vaartaja, O. 1960. Descriptions of new species of Rhizoctonia. Can. J. Bot. 38: 931-943. Saksena, H.K., and Vaartaja, O. 1961. Taxonomy, morphology and pathogenicity of Rhizoctonia species from forest nurseries. Can. J. Bot. 39: 627-647. Sneh, 8., Burpee, L., and Ogoshi, A. 1991. Identification of Rhizoc tonia species. The American Phytopathological Society, St Paul, Minn. Sutton, R.F. 1980. Root system morphogenesis. N.Z. J. For. Sci. 10: 264-292. Venn, K., Sandvik, M., and Langerud, B.R. 1986. Nursery routines, growth media and pathogens affect growth and root dieback in Norway spruce seedlings. Medd. Nor. Inst. Skogforsk. 39: 313-328. Warmbrodt, R.D., and Eschrich, W. 1985. Studies on the mycorrhizas of Pinus sylvestris L. produced in vitro with the basidiomycete Suillus variegatus (Sw. ex Fr.) O. Kuntze. 11. Ultrastructural as pects of the mycorrhizal rootlets. New Phytol. 100: 403-418. Wilcox, H. 1954. Primary organisation of active and dormant roots of noble fir, Abies procera. Am. J. Bot. 41: 812-821. Wilcox, H. 1968. Morphological studies of the root of red pine, Pinus resinosa. Am. J. Bot. 55: 247-254. © 1997 NRC Canada V 351 V.ll RHIZOCTONIA ASSOCIATED WITH FOREST TREES ARI M. HIETALA 1 AND ROBIN SEN 2 'Finnish Forest Research Institute, P. O. Box 18, FIN-01301 Vantaa, Findland and department of Biosciences, Division of General Microbiology, P.O. Box 56, FIN-00014 University of Helsinki, FINLAND I. Introduction Many Rhizoctonia species are economically important plant pathogens in agriculture and thus receive considerable attention. However, relatively little is known about these fungi and their effects on trees in forestry production. There are well documented cases of Rhizoctonia-hukcd diseases of tree seedlings cultivated in forest nurseries although information concerning their interactions with forest trees in natural ecosystems is limited and fragmentary. The early descriptions of Rhizoctonia spp. associated with tree species were mainly based on a few anamorphic traits. More recently, a re-evaluation of these species using presently accepted criteria (Parmeter and Whitney, 1970; Ogoshi, 1975) has resulted in the exclusion of many taxa from the genus Rhizoctonia (Andersen and Stalpers, 1994). In this review, the conventions of recently published checklists for Rhizoctonia are followed (Sneh et al., 1991; Andersen and Stalpers, 1994). Where possible, the present taxonomic position of the earlier described species will be indicated. II. Rhizoctonia in forest tree nurseries In a comprehensive nursery study, Saksena and Vaartaja (1961) grouped Rhizoctonia species into three classes that caused (a) root and hypocotyl rot and damping-off, (b) varying root disorders with intracellular chlamydospores (= monilioid hyphae) infections and (c) no apparent disease symptoms following root penetration. Later reports also confirmed their role in shoot diseases of trees. A. Rhizoctonia as a damping-off agent Damping-off, either as pre- or post-emergence is a common disease of seeds, germlings and young seedlings of many plants including woody species. It has long been known that anamorphs attributed to Rhizoctonia solani Kiihn (multinucleate, see Parmeter and Whitney, 1970) are a causal agent of damping-off in conifer seedlings (Wiant, 1929). World-wide, the seeds and young seedlings of a number of conifer and broad-leaved species (e.g. Pinus, Picea, Larix, Ulmus, Eucalyptus spp.) are known to be attacked (Hartley, 1921; Roth and Riker, 1943; Wright, 1944; Vaartaja and Cram, 1956; Saksena and Vaartaja 1960, 1961; Vaartaja et al., 1961; Vaartaja, 1967; Gomez-Nava, 1967; Sharma et al., 1984; Perrin and Sampangi, 1986). Another multinucleate species, R. endophytica var. filicata Saks, and Vaar (= R. zeae Voorhees) (see Sneh et al., 1991), was also reported as a damping-off pathogen on conifer seedlings (Saksena and Vaartaja 1961; Vaartaja, 1967) B. Sneh et ai. (eds.), Rhizoctonia Species: Taxonomy, Molecular Biology, Ecology, Pathology and Disease Constrol, 351-358 © 1996 Kluwer Academic Publishers. Printed in the Netherlands. AM HIETALA & R SEN 352 The anastomosis groups (AGs) (see Chapter 1.A2) of R. solani associated with trees have only been determined in a few studies. The most common, AG 4, has been isolated from Pinus banksiana Lamb, and Picea glauca Voss (Anderson, 1982), nursery soils (Camporota and Perrin, 1994) and the soil amendment, pine bark mulch (Huang and Kuhlman, 1990). Isolates from nursery soils, representing AGs 4 (the most common group), 5 and 1-2, but not AG 2-2, were very aggressive damping-off pathogens on Pinus nigra Arnold and the AG 4 isolate from pine bark mulch caused damping-off on Pinus elliottii Engelm. var. elliottii. Which AGs were involved in the early studies of Saksena and Vaartaja (1961)? The most common species, Ceratobasidium praticola (Kotila) Olive, causing damping-off, is presently included in the Thanatephorus cucumeris complex (and suggested to be T. praticola , Sneh et al ., 1991), despite sterigmatic characteristics previously regarded as specific, and now forms AG 4 of R. solani (Ogoshi, 1975). In examining the AG affinities of many earlier studied isolates, Parmeter et al. (1969) confirmed that three C. praticola isolates and one R. solani isolate (Saksena and Vaartaja, 1961), R. solani var. cedri deodarae (Castellani, 1934b) and five North American conifer isolates from different sources were all representatives of AG 4. This highlights the common occurrence of AG 4 on trees and seedlings. Binucleate Rhizoctonia spp. causing damping-off in conifer seedlings include R. callae Cast. (Saksena and Vaartaja, 1961), R. endophytica var. endophytica Saks, and Vaar. (= AG-A, see Ogoshi et al., 1983) (Saksena and Vaartaja, 1961; Gomez-Nava, 1967), and a Rhizoctonia sp. AG-E (=CAG 3, see Ogoshi et al., 1983) (Huang and Kuhlman 1990). The damping-off pathogens, Rhizoctonia, Pythium and Fusarium are übiquitous inhabitants in forest nursery soils (e.g. Perrin and Sampangi 1986) and reports indicate that seedlings grown directly in seed beds are more affected by damping-off than containerized seedlings that are normally cultivated in soil-free growth media. The activities of these pathogens are strongly dependent on prevailing soil conditions, e.g. pH, temperature, moisture etc. (Roth and Riker 1943; Camporota and Perrin 1994). Soil fumigation and seed treatments with fungicides have been recommended in nursery manuals as measures for broad-spectrum control. Potential damping-off pathogens, including R. solani (AG 4), may also be introduced into soils via organic amendments (Huang and Kuhlman, 1990). Huang and Kuhlman (1991) subsequently formulated a mixture of organic and inorganic materials which stimulated antagonistic fungi and reduced the populations of R. solani and Pythium aphanidermatum (Edson) Fitzp. enabling effective control of damping-off both in fumigated and non-fumigated soils. B. Rhizoctonia as a root pathogen of older seedlings Descriptions of Rhizoctonia spp. causing root damage in older conifer seedlings are mainly those of Saksena and Vaartaja (1960, 1961). Fungi were isolated from various conifer species displaying damping-off symptoms, mycorrhizal rootlets or apparently non-diseased seedlings. Isolates of R. solani, R. endophytica var endophytica (= AG-A), R. endophytica var. filicata (= R. zeae) and R. callae, causing damping-off, and R. repens Bernard (binucleate, see Sneh et al. 1991) also induced stunted shoot and root growth in seedlings of Pinus sylvestris L. , P. resinosa Ait., Ulmus americana L., Caragana arborescens Lam. and Thuja occidentalis L. A distinguishing characteristic of R. endophytica var. endophytica, R. callae and R. repens was the production of monilioid hyphae in root cortical cells of all test plants. RHIZOCTONIA ON FOREST TREES 353 However, the results obtained from these tests conducted under sterile conditions were only considered indicative of the potential pathogenicity of these isolates (Saksena and Vaartaja, 1961). More recently, root dieback of both containerized and bare-rooted Norway spruce [Picea abi.es L. (Karst.)] and Scots pine (P. sylvestris) seedlings has been a considerable problem in Nordic nurseries (Venn et al., 1986; Lilja et al., 1992). The visual symptoms: wilting of young shoots, hanging tops, retarded height growth, discoloration of the foliage and partial or total death of the root system often appear after midsummer, on first year seedlings or occasionally later during the second growing season. Surveys for fungi present in the diseased roots and pathogenicity testing suggest that root dieback is a complex phenomenon involving both Rhizoctonia spp. and Pythium spp. (Venn et al., 1986; Lilja et al., 1992). Pathogenicity experiments performed under nursery conditions implicated the supporting sand beds as an inoculum source for Rhizoctonia spp. in container seedling production (Venn et al., 1986). Based on conserved anamorphic and induced teleomorphic characteristics, the Rhizoctonia sp. causing damage on nursery tree stocks in Norway and Finland represents a single species possessing uninucleate cells (Hietala et al., 1994; see preface). Basidial characteristics refer it to the teleomorph genus Ceratobasidium, possibly C. bicorne Erikss. and Ryv., which is exceptional since all the known anamorphs of Ceratobasidium species are binucleate (see Sneh et al., 1991). However, there was no affinity with the other Ceratobasidium AG testers, and similar analysis with C. bicorne, only available as herbarium material, was not possible (Hietala et al., 1994). In pathogenicity tests with Norway spruce (Hietala, 1995) and Scots pine (Sen and Hietala, unpublished), uninucleate Rhizoctonia isolates attacked all parts of the root system, although the root tips were most heavily infected resulting in a stunted root system morphology (see preface). Cortical cells infected with monilioid cells were occasionally observed. In the Finnish surveys, non-pathogenic binucleate Rhizoctonia spp. were also isolated from the roots of similarly diseased and healthy-looking conifer seedlings (Lilja et al., 1992; Lilja 1994). In a case study, binucleate Rhizoctonia spp. were found to co-exist with the uninucleate Rhizoctonia sp. in the same root system and were divided into four anastomosis groups including AG-I of the genus Ceratobasidium and diree anastomosis groups not related to known anastomosis groups of Ceratobasidium (Hietala, 1995). In pathogenicity tests on Norway Spruce (Hietala, 1995) and Scots pine (Sen and Hietala, unpublished) seedlings, isolates representing these groups infected only basal root regions and cortical cells filled with monilioid hyphae were commonly observed. Although the infection had no effect on root growth of Norway spruce seedlings, isolates representing AG-I and two other AGs significantly increased shoot and root growth of Scots pine seedlings. A non orchid mycorrhizal relationship could be hypothesized with the latter conifer species (see chapters V.14 and VI. B4) although fungal infection levels were low (<2O % of root length infected). C. Other Rhizoctonia associated with tree roots Castellani (1934b) described three species, R. pini-insignis Cast., R. quercus Cast, and R. fraxini Cast. , from tree roots. TTie former two were respectively isolated from nursery grown Pinus insignis Dougl. (= Pinus radiata D. Don) and Quercus AM HIETALA & R SEN 354 pedunculata Ehrh. and the latter from apparently naturally growing Fraxinus excelsior L. Burpee et al. (1980) found R. fraxini and R. pini-insignis to be binucleate and R. quercus uninucleate although Ogoshi et al. (1983) could not confirm a uninucleate condition in the latter species. Unfortunately, the pathogenicity of these species was not tested on the respective hosts (Castellani 1934 a). D. Rhizoctonia associated with shoot diseases In India, R. solani has been identified as the causal agent of web blight in a number of nursery-grown broad-leaved trees including Eucalyptus , Acacia and Albizia species (Sharma et al., 1984; Sharma and Sankaran 1984; Sankaran et al., 1986; Mehrotra, 1990). The symptoms vary depending on the host, but a common feature is the premature defoliation of mycelium covered diseased leaves of young plants (Mehrotra, 1990). Isolates of R. solani obtained from the different hosts could be separated into three morphological groups, but cross inoculations of different hosts indicated no host specificity (Mehrotra, 1990). Another shoot disease caused by R. solani was reported in the same study; top flagging of khasi pine (Pinus kesiya Royle ex Gordon). Diseased nursery seedlings, 15 - 25 cm in height, displayed withered tops which later drooped and turn ash-colored. Infected needles were loosely bound with hyphae and the stems were also attacked. In both these shoot diseases, the respective host trees were most susceptible to R. solani over the wet season when the prevailing humid conditions enabled foliage colonization from soil inoculum sources via the stems (Mehrotra, 1990). Certain weed species were also highly susceptible to the disease and were suspected of being an additional source of infection. Disease control measures involve immediate removal of affected seedlings, weeding and a reduction in sowing densities. Needles of four- to five-year-old Norway spruce seedlings have been reported to be killed by a binucleate Rhizoctonia sp. in Norwegian forest nurseries (Roll-Hansen and Roll-Hansen, 1968). A binucleate Rhizoctonia sp. AG-E (=CAG 3), was shown to be associated with foliar blight of nursery grown longleaf pine (P. palustris Mill., English et al., 1986) and loblolly pine (P. taeda L., Runion and Kelly, 1993) in USA. On longleaf pine, the symptoms include necrosis of needles and terminal buds which are especially severe in sandy soils where sand accumulates around the needle bases and terminal buds. The sand possibly acts as an inoculum source and provides warm and humid conditions that apparently favour disease development. Affected needles of loblolly pine are characteristically bound in a mycelial webbing, turn grey and eventually drop-off. The severity of disease generally increases in seedbeds following crown closure which allow vegetative spread of the fungus via shoot contact (Runion and Kelly, 1993). Fungicide application does to some extent control the disease (Runion et al., 1994). Prior to sowing, seedbed soils are routinely fumigated with methyl bromide (English et al., 1986). III. Rhizoctonia in forest habitats Limited observations on the occurrence of the perfect states of Rhizoctonia confirm that the genus does thrive in forest habitats. Eriksson and Ryvarden (1973) reported the occurrence of Ceratobasidium cornigerum (Bourdot) Rogers on a variety of substrates and particularly the fresh bark of detached branches of both conifer and deciduous trees. Other Ceratobasidium species observed on bark of trees include C. RHIZOCTONIA ON FOREST TREES 355 pseudocornigerum M.P. Christ. (Eriksson and Ryvarden, 1973; Breitenbach and Kränzlin, 1986; Kotiranta and Saarenoksa, 1990) and C. stridii Erikss. and Ryv. (Eriksson and Ryvarden, 1973). The green needles of standing pines and the upper soil litter layer were implicated as a source of the orchid endophyte, R. goodyera repentis Costantin and Dufour (teleomorph, C. cornigerum ), in baiting experiments using the compatible orchid host (Downie, 1943). Understory plants have been found to host T. cucumeris (Poldmaa et al. 1982; Kotiranta and Saarenoksa, 1993) and C. bicorne (Eriksson and Ryvarden (1973). A. Rhizoctonia spp. as disease agents in forests The nuts of beech (Fagus sylvatica L.) have been reported to be attacked by R. solani in France (Perrin and Muller, 1979) and Germany (Dubbel, 1989). In Denmark, beechnuts are often similarly affected by Rhizoctonia (J. Koch, personal communication) and the isolates appear to be binucleate (Koch and Hietala, unpublished). The soilborne nature of R. solani was demonstrated in comparisons of seed from the forest floor with those collected from nets suspended above the ground (Dubbel, 1989). The disease is characterized by a decay of the cotyledon bearing tissue within the seed resulting in reduced germination or death after cotyledon emergence (Perrin and Muller, 1979). Higher disease incidence is related to increased pH and the level of soil organic material but soil cultivation before the mast is a good disease control strategy. It is recommended that beechnuts are rapidly harvested and where possible incubated before storage at 34 - 37 °C at 10 % relative humidity for 24 hours. This procedure significantly reduced disease incidence (Perrin and Muller, 1979). Surface sterilized seeds of Norfolk Island pine Araucaria heterophylla (Salisbury) Franco, growing in Egypt, were found to harbor Rhizoctonia solani while still within cones on the tree (El-Lakany et al., 1981). The fungus was isolated from the endosperm and embryo tissues and was also implicated as the principle seedborne pathogen causing pre- and post emergence damping-off in this and two other Araucaria species (Kamara et al., 1981). In two tropical Indian forests, R. solani has been identified as the causative agent of a root rot disease on seedlings of Phyllanthus emblica L. , Quercus serrata Thunb. and Citrus sp. (Chakravarty and Mishra, 1982). Disease symptoms, on mainly one to six-month-old seedlings, included either wilting or, in severe cases, stem collapse due to rotting of tissues at the base of the seedlings. The high temperatures and rainfall prevalent between May and August coincided with the highest disease incidence. IV. Future prospects A range of Rhizoctonia species are reported to be associated with trees both in nurseries and forest habitats. However, there is still a clear need for further identification of the putative R. solani anamorph. This was highlighted by Parmeter et al. (1967) who observed that mycelial and cultural characteristics of some Rhizoctonia species with a Ceratobasidium perfect state resemble those of R. solani so closely that only numbers of nuclei per cell or the perfect state distinguish them. Burpee et al. (1980) also stated that "many of the binucleate isolates in AGs-E -F and -R (=CAGs 3, 4 and 5) were morphologically indistinguishable from isolates of R. solani". Yet, in the majority of papers reviewed the observed anamorph was assigned to R. solani although neither the teleomorph nor the nuclear condition of cells were described. As induction of the teleomorph is problematic in many species, the nuclear condition is a 356 AM HIETALA & R SEN minimum diagnostic characteristic that can easily be determined using a number of nuclear stains (see Sneh et al., 1991). Few studies have been reported on a number of R. solani anastomosis groups (see Chapter 1.A2), particularly AG 4, that are associated with nursery tree seedlings. The AGs represent independent genetic units that often show different host preferences and levels of pathogenicity (see Chapter 11. 1). As the AG testers of R. solani and also Ceratobasidium are standardized and available from culture collections (e.g. American Type Culture Collection), the affinities of unknown multi- and bi-nucleate Rhizoctonia isolates associated with all plants, including trees, can be readily determined. This is of particular importance in enabling valid comparisons of the increasing data on the effects of Rhizoctonia spp. in nursery tree production and in natural forest ecosystems. It is clear that Rhizoctonia spp. are associated with forest trees but are mainly pathogens of young seedlings in forest nurseries. Increasing global pressure for re forestation will require expanded nursery seedling production where conventional chemical disease control practices have often proved ineffective. Due to their broad spectrum toxicity, the use of many of the pesticides and fumigants e.g. benomyl and methyl bromide is being phased-out leading the way to the development of biological control solutions. For the successful application of this alternative control strategy further detailed information is needed on the Rhizoctonia spp. involved in seedling diseases, their alternative host preferences (e.g. weed species) and interactions with other pathogens (e.g. Pythium and Fusarium spp.) and beneficial micro-organisms (e.g. Trichoderma spp. and mycorrhizal fungi, see chapters V 1.82, V 1.84 and V 1.87). Re-forestation of former agricultural land also may present additional problems relating to the potential broad host-range of pathogenic Rhizoctonia species. These challenges will require increased effort and co-operation between researchers in fields such as Rhizoctonia taxonomy and genetics, plant/tree physiology and pathology, agronomy, silviculture, molecular biology and biotechnology. V. Acknowledgements We thank Dr. Kari Korhonen for valuable comments on the manuscript and the financial support of Suomen Luonnonvarain Tutkimussäätiö and the Academy of Finland. VI. References Andersen TF & Stalpers JA (1994) A check-list of Rhizoctonia epithets. Mycotaxon 51:437-457. Anderson NA (1982) The genetics and pathology of Rhizoctonia solani. Annu. Rev. Phytopathol. 20:329- 347. Burpee LL, Sanders PL & Cole H Jr (1980) Anastomosis grouping among isolates of Ceratobasidium comigerum and related fungi. Mycologia 72:689-701. Breitenbach J & Kränzlin F (1986) Pilze der Schweiz. Band 2. Nichtblätterpilze. Verlag Mykologia, Luzern. Camporota P & Perrin R (1994) Survey for damping-off in forest nurseries in France. Preliminary results. 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Saksena HK & Vaartaja O (1960) Descriptions of new species of Rhizoclonia. Can. J. Bot. 38:931-943. Saksena HK & Vaartaja O (1961) Taxonomy, morphology and pathogenicity of Rhizoclonia species from forest nurseries. Can. J. Bot. 39:627-647. Sankaran KV, Balasundaran M & Sharma JK (1986) Seedling diseases of Azadirachla in Kerala, India. Eur. J. For. Path. 16:324-328. 358 AM HIETALA & R SEN Sharma JK & Sankaran KV (1984) Rhizoclonia web blight of Albizia falcalaria in India. Eur. J. For. Path. 14:261-264. Sharma JK, Mohanan C & Florence JM (1984) Nursery diseases of Eucalyptus in Kerala. Eur. J. For. Path. 14:77-89. Sneh B, Burpee L & Ogoshi A (1991) Identification of Rhizoclonia species. The APS Press, St. Paul Minnesota 133 pp.. Vaartiya O (1967) Damping-ofT pathogens in South Australian nurseries. Phytopathology 57:765-768. Vaartaja O & Cram WH (1956) Damping-off pathogens of conifers and of Caragana in Saskatchewan. Phytopathology 46:391-397. Vaartaja O, Cram WH & Morgan GA (1961) Damping-off etiology in forest nurseries. Phytopathology 51:35-42. Venn K, Sandvik M & Langerud BR (1986) Nursery routines, growth media and pathogens affect growth and root dieback in Norway spruce seedlings. Meddel. Norsk Inst. Skogforsk. 39:313-328. Wiant JS (1929) The Rhizoclonia solani damping-off of conifers, and its control by chemical treatments of the soil. N.Y. (Cornell) Agr. Exp. Sta. Mem. 124. 64 pp. Wright E (1944) Damping-off in broadleaf nurseries of the Great Plains region. J. Agric. Res. 69:77-94. ISBN 951-40-1617-3 ISSN 0358-4283 Hakapaino Oy 1998